http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Kazuko
2012.igem.org - User contributions [en]
2024-03-29T09:03:47Z
From 2012.igem.org
MediaWiki 1.16.0
http://2012.igem.org/Team:KIT-Kyoto/Home
Team:KIT-Kyoto/Home
2012-10-09T12:19:12Z
<p>Kazuko: </p>
<hr />
<div>{{KIT-Kyoto.Header}}<br />
{{KIT-Kyoto}}<br />
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<div id="HIDARI"><br />
<br><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/5/50/KITFUN.JPG" width="165" height="200"><br />
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<div id="MIGI"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6a/Wikitop写真.jpg" width="330" height="470" align="left" hspace="10"><br />
<div align="center"><font size="4">Drosophila Melanogaster Workshop</font></div><br />
<br><br />
<font line-height="15px"><I>Drosophila melanogaster</I> has been used for a genetic study as model organism for a long time and brought us much discovery. And we are sure that the benefit continues from now on.<br />
<br><br />
Therefore we KIT-Kyoto team aim at the production of the disease model Drosophila which expresses the responsible gene of MALT lymphoma that is one of leukemia that we were not able to achieve in a meeting of the last year. It is thought that we can contribute to elucidation of the mechanism of this disease and the development of the therapeutic drug by promoting this project.<br />
<br><br />
In addition, we think about what we can do in order to continue researches using Drosophila melanogaster in the world. The structure and behavior of the gene cluster are often found by pushing forward the study about genes systematically and cooperatively.<br />
<br><br />
So, this year we aim at the design of the parts with which a study that we use the Drosophila can expand in iGEM in future.<br />
<br><br />
If these projects are realized, the study using D. melanogaster will step forward to the new one step again.<br />
<br><br />
</font><br />
</div><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td ColSpan="2" width="965"><br />
<div id="NAKAMI"><br />
<br><br />
<div align="center"><br />
<font size="4">Our Sponsors</font><br />
<br><br><br />
<a href="http://www.kit.ac.jp/"><img src="https://static.igem.org/mediawiki/2012/e/ea/KITkyoto.png" width="100" height="100"></a> <a href="http://www.cosmobio.co.jp/"><img src="https://static.igem.org/mediawiki/2012/5/55/コスモ英語JPEG.jpg" width="140" height="100"></a><br />
<br><br />
<br><br />
<a href="http://www4.clustrmaps.com/user/6a8fdafd"><img src="http://www4.clustrmaps.com/stats/maps-no_clusters/2012.igem.org-Team-KIT-Kyoto-thumb.jpg" alt="Locations of visitors to this page" /><br />
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</div><br />
</div><br />
</td><br />
</tr><br />
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</body><br />
</html></div>
Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Home
Team:KIT-Kyoto/Home
2012-10-09T12:18:25Z
<p>Kazuko: </p>
<hr />
<div>{{KIT-Kyoto.Header}}<br />
{{KIT-Kyoto}}<br />
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<br><br />
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<div id="MIGI"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6a/Wikitop写真.jpg" width="330" height="470" align="left" hspace="10"><br />
<div align="center"><font size="4">Drosophila Melanogaster Workshop</font></div><br />
<br><br />
<font line-height="15px"><I>Drosophila melanogaster</I> has been used for a genetic study as model organism for a long time and brought us much discovery. And we are sure that the benefit continues from now on.<br />
<br><br />
Therefore we KIT-Kyoto team aim at the production of the disease model Drosophila which expresses the responsible gene of MALT lymphoma that is one of leukemia that we were not able to achieve in a meeting of the last year. It is thought that we can contribute to elucidation of the mechanism of this disease and the development of the therapeutic drug by promoting this project.<br />
<br><br />
In addition, we think about what we can do in order to continue researches using Drosophila melanogaster in the world. The structure and behavior of the gene cluster are often found by pushing forward the study about genes systematically and cooperatively.<br />
<br><br />
So, this year we aim at the design of the parts with which a study that we use the Drosophila can expand in iGEM in future.<br />
<br><br />
If these projects are realized, the study using D. melanogaster will step forward to the new one step again.<br />
<br><br />
</font><br />
</div><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td ColSpan="2" width="965"><br />
<div id="NAKAMI"><br />
<br><br />
<div align="center"><br />
<font size="4">Our Sponsors</font><br />
<br><br><br />
<a href="http://www.kit.ac.jp/"><img src="https://static.igem.org/mediawiki/2012/e/ea/KITkyoto.png" width="100" height="100"></a> <a href="http://www.cosmobio.co.jp/"><img src="https://static.igem.org/mediawiki/2012/5/55/コスモ英語JPEG.jpg" width="140" height="100"></a><br />
<br><br />
<br><br />
<a href="http://www4.clustrmaps.com/user/6a8fdafd"><img src="http://www4.clustrmaps.com/stats/maps-no_clusters/2012.igem.org-Team-KIT-Kyoto-thumb.jpg" alt="Locations of visitors to this page" /><br />
<a href="http://s11.flagcounter.com/more/HZGk"><img src="http://s11.flagcounter.com/count/HZGk/bg_FFFFFF/txt_000000/border_FFFFFF/columns_6/maxflags_12/viewers_0/labels_0/pageviews_1/flags_0/" alt="free counters" border="0"></a><br />
</div><br />
</div><br />
</td><br />
</tr><br />
</div> <br />
</table><br />
</body><br />
</html></div>
Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Home
Team:KIT-Kyoto/Home
2012-10-09T12:17:45Z
<p>Kazuko: </p>
<hr />
<div>{{KIT-Kyoto.Header}}<br />
{{KIT-Kyoto}}<br />
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<head><br />
<script style="text/css"><br />
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float:left; <br />
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<br><br />
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<img src="https://static.igem.org/mediawiki/2012/5/50/KITFUN.JPG" width="165" height="220"><br />
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<br><br />
<br><br />
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</td><br />
<br />
<td width="790px" height="520px" valign="top"><br />
<div id="MIGI"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6a/Wikitop写真.jpg" width="330" height="470" align="left" hspace="10"><br />
<div align="center"><font size="4">Drosophila Melanogaster Workshop</font></div><br />
<br><br />
<font line-height="15px"><I>Drosophila melanogaster</I> has been used for a genetic study as model organism for a long time and brought us much discovery. And we are sure that the benefit continues from now on.<br />
<br><br />
Therefore we KIT-Kyoto team aim at the production of the disease model Drosophila which expresses the responsible gene of MALT lymphoma that is one of leukemia that we were not able to achieve in a meeting of the last year. It is thought that we can contribute to elucidation of the mechanism of this disease and the development of the therapeutic drug by promoting this project.<br />
<br><br />
In addition, we think about what we can do in order to continue researches using Drosophila melanogaster in the world. The structure and behavior of the gene cluster are often found by pushing forward the study about genes systematically and cooperatively.<br />
<br><br />
So, this year we aim at the design of the parts with which a study that we use the Drosophila can expand in iGEM in future.<br />
<br><br />
If these projects are realized, the study using D. melanogaster will step forward to the new one step again.<br />
<br><br />
</font><br />
</div><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td ColSpan="2" width="965"><br />
<div id="NAKAMI"><br />
<br><br />
<div align="center"><br />
<font size="4">Our Sponsors</font><br />
<br><br><br />
<a href="http://www.kit.ac.jp/"><img src="https://static.igem.org/mediawiki/2012/e/ea/KITkyoto.png" width="100" height="100"></a> <a href="http://www.cosmobio.co.jp/"><img src="https://static.igem.org/mediawiki/2012/5/55/コスモ英語JPEG.jpg" width="140" height="100"></a><br />
<br><br />
<br><br />
<a href="http://www4.clustrmaps.com/user/6a8fdafd"><img src="http://www4.clustrmaps.com/stats/maps-no_clusters/2012.igem.org-Team-KIT-Kyoto-thumb.jpg" alt="Locations of visitors to this page" /><br />
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</div><br />
</div><br />
</td><br />
</tr><br />
</div> <br />
</table><br />
</body><br />
</html></div>
Kazuko
http://2012.igem.org/File:KITFUN.JPG
File:KITFUN.JPG
2012-10-09T12:14:06Z
<p>Kazuko: </p>
<hr />
<div></div>
Kazuko
http://2012.igem.org/Team:KIT-Kyoto
Team:KIT-Kyoto
2012-09-27T02:41:30Z
<p>Kazuko: </p>
<hr />
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<br><br />
<font color=white size="5"><a href="https://2012.igem.org/Team:KIT-Kyoto/Home">>>ENTER</a></font><br />
<BR><br />
<font color=gray>This movie requires the latest version of <a href="http://www.adobe.com/go/getflashplayer" target="_blank" border="0">Adobe Flash Player</a>.</font><br />
<br><br />
<font color=gray size=1><i>Copyright © 2012 <a HREF="https://2012.igem.org/Main_Page">iGEM</a>, <a href="https://2012.igem.org/Team:KIT-Kyoto/Home">KIT-Kyoto</a> All Rights Reserved.</i></font><br />
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<br><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto
Team:KIT-Kyoto
2012-09-27T02:41:12Z
<p>Kazuko: </p>
<hr />
<div><html><br />
<head><br />
<meta http-equiv="Refresh" content="30"URL="https://2012.igem.org/Team:KIT-Kyoto"><br />
<style type="text/css"><br />
<br />
/*配置 :)*/<br />
#NAKAMI {<br />
background:transparent url(https://static.igem.org/mediawiki/2012/e/e2/Khaikei3.PNG);<br />
width: 925px;<br />
padding: 20px;<br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week7
Team:KIT-Kyoto/Notebook-week7
2012-09-27T02:38:50Z
<p>Kazuko: </p>
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<h2>September 18th</h2><br />
<br><br />
<br />
By red eye screening, we identified the strain 7 and strain 10 to be the successfully transformed lines. Photographs for the transformed red (yellow) eye fly (strain 10) were taken.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/c/c1/HAEE2.JPG" width="320" height="220"><br />
<br><br><br />
<br />
<h2>September 19th</h2><br />
<br><br />
<br />
By red eye screening, we identified the strain 13 and strain 14 to be the successfully transformed lines. Photographs for the transformed red (orange) eye fly (strain 13) were taken.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/a/a6/Strain13.JPG" width="320" height="220"><br />
<br><br><br />
Drosophila S2 cells (2.5 X 10<sup>5</sup> cells per well) were plated on the 24 well plate and cultured in the Schneider’s Drosophila medium containing 10% fetal bovine serum at 25 ℃.<br />
<br><br><br />
<br />
<h2>September 20th</h2><br />
<br><br />
By red eye screening, we identified the strain 23 and strain 29 to be the successfully transformed lines.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 21st</h2><br />
<br><br />
Red eye screening was continued.<br />
<br><br><br />
<br />
<br />
<h2>September 22nd</h2><br />
<br><br />
Red eye screening was continued.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<h2>September 23rd</h2><br />
<BR><br />
By red eye screening, we identified the strain 56 and strain 65 to be the successfully transformed lines. These transformed flies are kept at 25℃ and later will be further inspected for chromosome linkage of the transgene. <br />
<br><br><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Humanpractice
Team:KIT-Kyoto/Humanpractice
2012-09-27T00:56:41Z
<p>Kazuko: </p>
<hr />
<div>{{KIT-Kyoto}}<br />
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<h2>Introduction</h2><br />
<br><br />
People all over the world today tend to lose interest in science, so we think that this tendency disturb progress of synthetic biology. For example, ordinary people seem to think recombinant DNA technologies, which are the basis of synthetic biology as unpleasant. In this way, they do not fully understand what recombinant DNA technologies are, and they still hesitate to buy and eat GM foods. These problems seem to be faced in the world when recombinant DNA technologies will be developed and used in many fields. We want a lot of people to know not only in Japan but also in the world, so we try these things as Human Practice.<br />
<br><br><br />
<h2>At the open campus (2012/08/10,11)</h2><br />
<br><br />
We prepared a booth to introduce iGEM in KIT Open Campus and explained our activity of iGEM to a visitor.<br />
<br><br />
To make the presentation simple, we tried to avoid using technical terms.<br />
<br><br />
Besides, we displayed model organisms used in genetics, such as fruit-fly Drosophila melamogaster, baker's yeast Saccharomyces cereviciae, and colon bacterium Eschericia coli.<br />
Students could see Drosophila mutants. And we displayed "E.Coli Pen" and some pictures drawn with it.<br />
<br><br />
We aimed to make their understanding clear, and make them feel familiar to genetic engineering.<br />
<br><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6e/P1040278.JPG" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/7/77/NEC_0492.JPG" width="200" height="200" > <img src="https://static.igem.org/mediawiki/igem.org/8/8c/P1040255.JPG" width="200" height="200" > <img src="https://static.igem.org/mediawiki/2012/1/17/NEC_0478.JPG" width="200" height="200" ><br />
<br><br />
<br><br><br />
<h2>Survey</h2><br />
<br><br />
Because we wanted to know what kind of image does a thousand and more people of the age to reach the elderly from a high school student have to recombinant DNA technologies, Drosophila melanogaster and Leukemia which we use in Drosophila melanogaster Workshop. For this purpose, we KIT-Kyoto sent out questionnaire survey to them in cooperation with team <a href="https://2012.igem.org/Team:Osaka">Osaka</a> and <a href="https://2012.igem.org/Team:Ehime-Japan">Ehime-Japan</a>. We collected answers from not only students major in biology but also in chemistry, information technology, mechanical engeneering and art in college, in addition to the attendee to the college's open day or people at facebook. Therefore, we could get various ways of thinking to synthetic biology, regardless of their age, job or majors in college.<br />
<br><br><br />
You can see our questionnaire <a href="https://static.igem.org/mediawiki/2012/8/8b/完成filekit.pdf">here</a>.<br />
<br><br />
Also, this is the <a href="https://static.igem.org/mediawiki/2012/8/82/完成Qkit.pdf">result</a>.<br />
<br><br />
<br><br />
The result says, 97% of people feel Drosophila as dirty, 81% feel as harmful. What does this mean? A lot of people confuse Drosophila and fly. Fly you find in your house is harmful, however, Drosophila can be used for gene therapy. So, it is very useful for us to study.<br />
<br><br />
Also, we asked about the therapy of leukemia. From the result of this survey, we found that people wants to use inexpensive and recurrence preventing drugs.<br />
<br><br><br />
<br />
<br />
<h2>Introduction of the class of Bioart (2012/09/19-21)</h2><br />
In our college KIT, bioart was firstly introduced as a lecture with practical course called‘Fusion of Science and Art Ⅰ using our project two years ago, E. coli pen in last year. Undergraduate students who major not only biology but also various fields of science can take the lecture.<br />
<br><br />
Students learned the mechanism that the ink of E. coli pen emits fluorescence and drew paintings on agar plates using E. coli inks. And they gave a presentation on their concept of bioart.<br />
<br><br />
Furthermore, the bioart described in E.coli Pen is carried by the textbook of the high school student.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/a/a8/BIOARTb.jpg" width="200" height="200" ><img src="https://static.igem.org/mediawiki/2012/4/43/BIOARTa.jpg" width="200" height="200"><img src="https://static.igem.org/mediawiki/2012/5/5a/KITBIO.JPG" width="200" height="200" > <img src="https://static.igem.org/mediawiki/2012/f/f7/大腸菌インク.jpg" width="300" height="200"><br />
<br><br><br />
<h2>Movie</h2><br />
According to the questionnaire, which we conducted, people think that genetic engineering has risk for environment and humans, and these are very complicated. And, they think Drosophila melanogaster, which we used in our experiments, is a harmful insect. In order to change the idea, we made a movie, which explains genetic engineering, and shows the significant usefulness of Drosophila melanogaster. This movie includes the scene of our lab work. By watching this movie, many people can get a correct knowledge of genetic engineering and Drosophila melanogaster, and change the idea. We hope people in the world would be interested in “science”.<br />
<br><br />
<br><br />
<div align="center"><iframe width="560" height="315" src="http://www.youtube.com/embed/ZpwVDHuDB-0?rel=0" frameborder="0" allowfullscreen></iframe><br />
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Kazuko
http://2012.igem.org/File:%E5%AE%8C%E6%88%90filekit.pdf
File:完成filekit.pdf
2012-09-27T00:56:21Z
<p>Kazuko: </p>
<hr />
<div></div>
Kazuko
http://2012.igem.org/File:%E5%AE%8C%E6%88%90Qkit.pdf
File:完成Qkit.pdf
2012-09-27T00:53:50Z
<p>Kazuko: </p>
<hr />
<div></div>
Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week1p
Team:KIT-Kyoto/Notebook-week1p
2012-09-26T13:42:45Z
<p>Kazuko: </p>
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<td width="800px" height="510px" valign="top"><br />
<div id="MIGI"><br />
<br />
<h2>First</h2><br />
<br><br />
The first number 1 indicates experiments about UAS.<br />
<br><br />
The first number 2 indicates experiments about GAL4.<br />
<br><br />
Second number 1 indicates making two experiments at the same time when we carried out ligation.<br />
<br><br />
Second number 2 indicates making three experiments at the same time when we carried out ligation.<br />
<br><br />
The parts we are going to submit are these seven, pSB1C3-UAS, pSB1C3-UAS-LacZ, pSB1C3-UAS-EGFP, pSB1C3-UAS-TNFAIP3,pSB1C3-GAL4, pSB1C3-HS-GAL4, and pSB1C3-Act5c-GAL4.<br />
<br><br><br />
<br />
<h2>August 23rd</h2><br />
<br><br />
<strong>1-1-1 and 2-1-1 Transformation of E. coli with pEGFP-C2 DNA and pAct5C-GAL4 DNA</strong><br />
<br><br />
E. coli transformed with pEGFP-C2 was spread on the LB Kanamycin(+) plate and that transformed with pAct5C GAL4 was spread on the LB ampicillin(+) plate.<br />
<br />
<br><br><br />
<br />
<br />
<h2>August 24th</h2><br />
<br><br />
<strong>1-1-2 and 2-1-2</strong><br />
<br><br />
The appropriate colonies were picked up and cultured in the LB medium containing the appropriate antibiotics.<br />
<br><br><br />
<br />
<br />
<h2>August 25th</h2><br />
<br><br />
<strong>1-1-3 and 2-1-3 Purification of the pEGFP-C2 DNA and the pAct5C-GAL4 DNA</strong><br />
<br><br />
These plasmid DNAs were purified from E. coli by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
<strong>1-1-4 Digestion of pEGFP-C2 DNA with BamHⅠ and BglⅡt</strong><br />
<br><br />
Restriction enzyme digestion was carried out in the following reaction<br />
<br><br><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Total DNA amount </Td><Td> 500ng </Td><Td> 1ug </Td></Tr><br />
<tr><td> pEGFP-C </td><td> 3uL </td><Td> 6uL </Td></tr><br />
<Tr><Td> H buffer(TOYOBO) </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> BamHⅠ(TOYOBO) </td><td> 1uL </td><Td> 1uL </Td></tr><tr><br />
<td> BglⅡ(TOYOBO) </td><td> 1uL </td><Td> 1uL </Td></tr><tr><br />
<td> 100×BSA </td><td> 0.5uL </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 39.5uL </td><Td> 36.5uL </Td></tr><tr><br />
<td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><br><br />
<strong>1-1-5 Agarose gel electrophoresis of the digested DNA </strong><br />
<br><br />
The digested DNA was applied to the agarose gel electrophoresis and linearized DNA was purified by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photograph of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d0/0825kit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-1-6 Digestion of pSB1C3DNA with EcoRⅠ and SpeⅠ</strong><br />
<br><br />
pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ at 37℃ for 16 hours.<br />
<br><BR><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA sample </td><td> 23uL </td></tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<tr><td> EcoRⅠ-HF(NEB) </td><td> 1uL </td></tr><tr><br />
<td> SpeⅠ(NEB) </td><td> 1.5uL </td></tr><br />
<td> 100×BSA </td><td> 0.5uL </td></tr><br />
<td> dH<sub>2</sub>O </td><td> 19uL </td></tr><br />
<td> Total <td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
<br />
<strong>1-1-7 PCR amplification of the UAS region and pSB1C3 DNA</strong><br />
<br><br />
DNA fragments containing UAS was amplified from the pUAST DNA and the BglⅡ site-containing pSB1C3 was also amplified by PCR.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA template </td><td> 0.5uL </td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 10uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 10uL </td></tr><tr><br />
<td> 25mM MgSO4 </td><td> 3.2uL </td></tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<td> KOD plus </td><td> 2uL </td></tr><br />
<td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<td> Total <td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Circle </Td></Tr><br />
<tr><td> 95℃</td><td> 2min</td><Td></Td></tr><br />
<Tr><Td> 95℃</Td><Td> 15sec</Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃</td><td> 30min(UAS) or 2min10sec(pSB1C3)</td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃</td><td> 2min30sec</td><Td> 25 cycle </Td></tr><br />
<td> 68℃</td><td> 2min30sec</td><Td></Td></tr><br />
<td> 14℃</td><td> ∞</td><Td></Td></tr><br />
</Table><br />
<br><br><br />
<br />
<br />
<br />
<br />
<br />
<h2>August 26th</h2><br />
<br><br />
<strong>1-1-8 Purification of the digested pSB1C3</strong><br />
<br><br />
pSB1C3 DNA double-digested with EcoRⅠ and SpeⅠ(prepared on 8/25) was applied to the agarose gel electrophoresis. The DNA fragment of interest was purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/0826.png" width="500" height="300"><br />
<br><br />
<br><br />
<h2>August 27th</h2><br />
<br><br />
<strong>1-1-9 Removing the multi-cloning site of the pEGFP-C2</strong><br />
<br><br />
pEGFP-C2 DNA digested with BglⅡ and BamHⅠwas self ligated in the following reaction.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pEGFP-C2(cut) </Td><Td> 1uL </Td></Tr><br />
<tr><td> Ligation high </td><td> 2.5uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 5uL </td></tr><tr><br />
<td> Total <td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-10</strong><br />
<br><br />
The ligated products were transformed into the E. coli XL-1 blue and the E. coli was apreaded on the LB Kanamycin(+) plate and cultured at 37℃ for 16 hours.<br />
<br><br><br />
<strong>1-1-11 Agarose gel electrophoresis of the DNA fragments containing UAS and the PSR products from the pSB1C3 </strong><br />
<br><br />
PCRproducts prepared on August 25th were applied to the agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/e6/0826bkit.png" width="500" height="300"><br />
<br><br><br />
Since the expected DNA fragments were detected, DNA was purified from the agarose gel by QIA quick Gel Extraction Kit. The purified DNAs were applied to the agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/99/0826ckit.png" width="500" height="300"><br />
<br><br><br />
<br />
<br />
<br />
<br />
<h2>August 28th</h2><br />
<br><br />
<strong>1-1-12 Single colony isolation from the E. coli transformed with pEGFP-C2 DNA</strong><br />
<br><br />
Single colony isolationwas carried out from the plate prepared on 8/27and cultured in 2.5mL LB Kanamycin(+) medium at 37℃ for 16 hours.<br />
<br><br><br />
<strong>2-1-4 Transformation of E. coli with pGaTB DNA or pAct5C-GAL4 DNA</strong><br />
<br><br />
E. coli Xl-1 blue was transformed with pGaTBDNA or pAct5C-GAL4 DNA and cultured on LB ampicillin(+) plate for 16 hours at 37℃.<br />
<br><br><br />
<br />
<br />
<br />
<h2>August 29th</h2><br />
<br><br />
<strong>2-1-5 Single colony isolation from the E. coli transformed with pGaTB DNA or pAct5C-GAL4 DNA</strong><br />
<br><br />
From the plate prepared on 8/28 single colonies were isolated and cultured in 2.5mL LB ampicillin(+) medium for 16 hours at 37℃.<br />
<br><br><br />
<strong>1-1-13 Purification of pEGFP-C2 DNA</strong><br />
<br><br />
pEGFP-C2 DNA was purified from E. coli prepared on 8/28 by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
<strong>1-1-14 Cut check of the pEGFP-C2 DNA</strong><br />
<br><br />
The purified pEGFP-C2(1-1-13) was digested with XhoⅠ and PstⅠ in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pEGFP-C2-1, -2, -3, -4 </Td><Td> 1uL </Td></Tr><br />
<tr><td> 10×H buffer(TOYOBO) </td><td> 0.5uL </td></tr><tr><br />
<tr><td> XhoⅠ </td><td> 0.1uL </td></tr><tr><br />
<tr><td> PstⅠ </td><td> 0.1uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 3.3uL </td></tr><tr><br />
<td> Total <td> 5uL </td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were applied to the agarose gel electrophoresis.as shown below. <br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/97/0828kit.png" width="500" height="300"><br />
<br><br><br />
Results: The DNAs were undigested by these enzymes, confirming that the multi-cloning site of the pEGFP-C2 was successfully removed.<br />
<br><br><br />
<strong>1-1-15 PCR amplification of the DNA fragments containing UAS and EGFP</strong><br />
<br><br />
Since we found that the previously used primers for PCR were wrong, PCR amplification of the DNA fragments containing UAS and EGFP region were carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 10uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 10uL </Td></Tr><br />
<Tr><Td> 25mM MgSO4 </Td><Td> 3.2uL </Td></Tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<Tr><td> KOD plus </td><td> 2uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<Tr><td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 1min(EGFP) </td><Td> 25 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 1min(EGFP) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
Agarose gel electrophoresis of the PCR products<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d2/0829b.png" width="500" height="300"><br />
<br><br><br />
Results: DNA fragments containing EGFP were successfully amplified, but not for the UAS.<br />
<br><br><br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week2p">>>>>>>>>>WEEK2</a></div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Humanpractice
Team:KIT-Kyoto/Humanpractice
2012-09-26T13:36:46Z
<p>Kazuko: </p>
<hr />
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<h2>Introduction</h2><br />
<br><br />
People all over the world today tend to lose interest in science, so we think that this tendency disturb progress of synthetic biology. For example, ordinary people seem to think recombinant DNA technologies, which are the basis of synthetic biology as unpleasant. In this way, they do not fully understand what recombinant DNA technologies are, and they still hesitate to buy and eat GM foods. These problems seem to be faced in the world when recombinant DNA technologies will be developed and used in many fields. We want a lot of people to know not only in Japan but also in the world, so we try these things as Human Practice.<br />
<br><br><br />
<h2>At the open campus (2012/08/10,11)</h2><br />
<br><br />
We prepared a booth to introduce iGEM in KIT Open Campus and explained our activity of iGEM to a visitor.<br />
<br><br />
To make the presentation simple, we tried to avoid using technical terms.<br />
<br><br />
Besides, we displayed model organisms used in genetics, such as fruit-fly Drosophila melamogaster, baker's yeast Saccharomyces cereviciae, and colon bacterium Eschericia coli.<br />
Students could see Drosophila mutants. And we displayed "E.Coli Pen" and some pictures drawn with it.<br />
<br><br />
We aimed to make their understanding clear, and make them feel familiar to genetic engineering.<br />
<br><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6e/P1040278.JPG" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/7/77/NEC_0492.JPG" width="200" height="200" > <img src="https://static.igem.org/mediawiki/igem.org/8/8c/P1040255.JPG" width="200" height="200" > <img src="https://static.igem.org/mediawiki/2012/1/17/NEC_0478.JPG" width="200" height="200" ><br />
<br><br />
<br><br><br />
<h2>Survey</h2><br />
<br><br />
Because we wanted to know what kind of image does a thousand and more people of the age to reach the elderly from a high school student have to recombinant DNA technologies, Drosophila melanogaster and Leukemia which we use in Drosophila melanogaster Workshop. For this purpose, we KIT-Kyoto sent out questionnaire survey to them in cooperation with team <a href="https://2012.igem.org/Team:Osaka">Osaka</a> and <a href="https://2012.igem.org/Team:Ehime-Japan">Ehime-Japan</a>. We collected answers from not only students major in biology but also in chemistry, information technology, mechanical engeneering and art in college, in addition to the attendee to the college's open day or people at facebook. Therefore, we could get various ways of thinking to synthetic biology, regardless of their age, job or majors in college.<br />
<br><br><br />
You can see our questionnaire <a href="https://static.igem.org/mediawiki/2012/3/35/The_Questionnair_KIT.pdf">here</a>.<br />
<br><br />
Also, this is the <a href="https://static.igem.org/mediawiki/2012/c/c2/QUESTIONNAIRE.RESULTS.pdf">result</a>.<br />
<br><br />
<br><br />
The result says, 97% of people feel Drosophila as dirty, 81% feel as harmful. What does this mean? A lot of people confuse Drosophila and fly. Fly you find in your house is harmful, however, Drosophila can be used for gene therapy. So, it is very useful for us to study.<br />
<br><br />
Also, we asked about the therapy of leukemia. From the result of this survey, we found that people wants to use inexpensive and recurrence preventing drugs.<br />
<br><br><br />
<br />
<br />
<h2>Introduction of the class of Bioart (2012/09/19-21)</h2><br />
In our college KIT, bioart was firstly introduced as a lecture with practical course called‘Fusion of Science and Art Ⅰ using our project two years ago, E. coli pen in last year. Undergraduate students who major not only biology but also various fields of science can take the lecture.<br />
<br><br />
Students learned the mechanism that the ink of E. coli pen emits fluorescence and drew paintings on agar plates using E. coli inks. And they gave a presentation on their concept of bioart.<br />
<br><br />
Furthermore, the bioart described in E.coli Pen is carried by the textbook of the high school student.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/a/a8/BIOARTb.jpg" width="200" height="200" ><img src="https://static.igem.org/mediawiki/2012/4/43/BIOARTa.jpg" width="200" height="200"><img src="https://static.igem.org/mediawiki/2012/5/5a/KITBIO.JPG" width="200" height="200" > <img src="https://static.igem.org/mediawiki/2012/f/f7/大腸菌インク.jpg" width="300" height="200"><br />
<br><br><br />
<h2>Movie</h2><br />
According to the questionnaire, which we conducted, people think that genetic engineering has risk for environment and humans, and these are very complicated. And, they think Drosophila melanogaster, which we used in our experiments, is a harmful insect. In order to change the idea, we made a movie, which explains genetic engineering, and shows the significant usefulness of Drosophila melanogaster. This movie includes the scene of our lab work. By watching this movie, many people can get a correct knowledge of genetic engineering and Drosophila melanogaster, and change the idea. We hope people in the world would be interested in “science”.<br />
<br><br />
<br><br />
<div align="center"><iframe width="560" height="315" src="http://www.youtube.com/embed/ZpwVDHuDB-0?rel=0" frameborder="0" allowfullscreen></iframe><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week4p
Team:KIT-Kyoto/Notebook-week4p
2012-09-26T13:32:36Z
<p>Kazuko: </p>
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<h2>September 13th</h2><br />
<br><br />
<strong>1-1-50 and 2-2-8 Isolating a single colony of E. coli carrying the candidate pSV1C3-UAS-LacZ or pSB1C3-HS-GAL4</strong><br />
<br><br />
We isolated colonies (one for pSB1C3-UAS-LacZ ,three for pSB1C3-HS-G4L4) and cultured in liquid medium(2.5ml LB Chloramphenicol(+)) at 37℃ for 16 hours. No transformed colony was detected for E. coli carrying the candidate pSB1C3-Act5C-GAL4.<br />
<br><br><br />
<strong>1-2-8 Cut check for the candidate pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP</strong><br />
<br><br />
The candidate pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP DNAs (made 9/12) were digested with EcoRⅠ/ SpeⅠ and BglⅡ, respectively.<br />
<br><br><br />
EcoRⅠand SpeⅠ<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA sample </Td><Td> 1uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 0.5uL </Td></tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.2uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.2uL </Td></Tr><Tr><br />
<Td> 100×BSA </Td><Td> 0.05uL </Td></Tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 3.25uL </Td></tr><tr><br />
<td> Total </td><Td> 5uL </Td></tr><br />
</Table><br />
<BR><BR><br />
BglⅡ<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA sample </Td><Td> 1uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><Td> 0.5uL </Td></tr><br />
<Tr><Td> BglⅡ </Td><Td> 0.2uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><Td> 3.3uL </Td></tr><br />
<td> Total </td><Td> 5uL </Td></tr><br />
</Table><br />
<br><br><br />
Then the digested samples were applied to Agarose gel electrophoresis in the order of no cut sample, EcoRⅠ/SpeⅠ-digested sample and BglⅡ-digested sample.<br />
<br><br><br />
Result of electrophoresis<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/3/3f/0913kit.png" width="500" height="300"><br />
<br><br><br />
Results: No insert DNA was detected. Therefore we were not successful for these cloning.<br />
<br><br><br />
<strong>1-1-51, 2-1-37 and 2-2-9. Ligation</strong><br />
<br><br />
Ligation of DNA fragments carrying GAL4 to pSB1C3 DNA was carried out for 2 hours at 16℃ in the reaction described below.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (double digested with BglⅡand SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (double digested with BglⅡand SpeⅠ) </td><Td> 1.5uL </Td></tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 2.5uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 2.5uL </Td></tr><tr><br />
<td> Total </td><Td> 7.5uL </Td></tr><br />
</Table><br />
<br><BR><br />
Ligation of DNA fragments carrying G4L4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to SB1C3 DNA was carried out for 1 hours at 16℃ in the reaction described below.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3(PCR)(double digested with XbaⅠand SpeⅠ) </Td><Td> 2.5uL </Td></Tr><br />
<tr><td> GAL4(double digested with BglⅡand SpeⅠ) </td><Td> 2.5uL </Td></tr><br />
<tr><td> HS or Act5c fragments (double digested with XbaⅠand BamHⅠ) </td><Td> 2uL </Td></tr><br />
<td> Ligation high </td><Td> 7uL </Td></tr><tr><br />
<td> Total </td><Td> 14uL </Td></tr><br />
</Table><br />
<br><BR><br />
Ligation of DNA fragments carrying EGFP or LacZ to pSB1C3-UAS was carried out for 1 hours at 16℃ in the reaction described below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS (double digested with BglⅡand SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> EGFP fragments or LacZ fragments (double digested with BglⅡand SpeⅠ) </td><Td> 2uL </Td></tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 2uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 5uL </Td></tr><tr><br />
<td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-52, 2-1-38 and 2-2-10 Transformation of E. coli by Ligation products</strong><br />
<br><br />
We did transformation of E. coli Xl-1 blue with each of ligation products. Finally, we spread them on the LB Chloramphenicol(+) plate and cultured at 37℃ for 16 hours.<br />
<br><br><br />
<br />
<h2>September 14th</h2><br />
<br><br />
<strong>1-1-53 and 2-1-39 Isolating a single colony of E. coli transformed with Ligation products</strong><br />
<br><br />
We picked up three colonies of E. coli carrying the candidate pSB1C3-G4L4, two from the candidate pSB1C3-UAS-EGFP and three from the candidate pSB1C3-UAS-LacZ (made on 9/13), and cultured in 2.5mL LB Chloramphenicol(+) liquid medium at 37℃ for 16 hours.<br />
<br><br><br />
<strong>1-1-54 and 2-2-11 Purification of the candidate pSB1C3-UAS-LacZ and pSB1C3-HS-GAL4 DNA</strong><br />
<br><br />
The pSB1C3-UAS-LacZ DNA and pSB1C3-HS-G4L4 DNA was purified from E. coli (cultivated on 9/12) by QIA prep Spin Miniprep Kit. <br />
<br><br><br />
<strong>1-1-55 and 2-2-12 Cut check of the candidate pSB1C3-UAS-LacZ, pSB1C3-UAS-EGFP, and pSB1C3-HS-GAL4 DNA</strong><br />
<br><br />
These candidate pSB1C3-UAS-EGFP DNA (prepared on 9/12), pSB1C3-UAS-LacZ DNA (prepared on 9/12), pSB1C3-UAS-LacZ DNA (prepared on 9/14) and pSB1C3-HS-G4L4 DNA (prepared on 9/14)<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA sample </Td><Td> 1uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 1uL </Td></tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.2uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.2uL </Td></Tr><Tr><br />
<Td> 100×BSA </Td><Td> 0.1uL </Td></Tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 7.5uL </Td></tr><tr><br />
<td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><br><br />
Digested samples were applied to the Agarose gel electrophoresis.<br />
<br><br />
From the left side,<br><br />
Two pSB1C3-UAS-EGFP DNA isolated from independent colonies (9/12) uncut , digested two pSB1C3-UAS-EGFP DNA (9/12),<br />
<br><br />
pSB1C3-UAS-LacZ DNA (9/12) uncut, digested pSB1C3-UAS-LacZ DNA (9/12)cut,<br />
<br><br />
pSB1C3-UAS-LacZ DNA (9/14) uncut, digested pSB1C3-UAS-LacZ DNA (9/14), <br />
<BR><br />
pSB1C3-HS-G4L4 DNA from three independent colonies (9/14) uncut, digested pSB1C3-HS-G4L4 DNA from three independent colonies (9/14)<br />
<br><br><br />
<br />
Results<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/ec/0914kit.png" width="500" height="300"><br />
<br><br><br />
We found that pSB1C3-UAS-LacZ DNA (9/14) was successfully constructed !. This is the first one we successfully constructed as a Biobrick part.<br />
<br><br><br />
<strong>2-2-13 Ligation</strong><br />
<br><br />
Ligation of DNA fragments carrying G4L4 and those carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA was carried out for 2 hours at 16℃ in a reaction described below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3(PCR) (double digested with XbaⅠand SpeⅠ) </Td><Td> 2uL </Td></Tr><br />
<tr><td> GAL4 (double digested with BglⅡand SpeⅠ) </td><Td> 3uL </Td></tr><br />
<tr><td> HS fragments or Act5c fragments (double digested with XbaⅠand BamHⅠ) </td><Td> 1uL(HS) or 2.5uL(Act5c) </Td></tr><tr><br />
<td> Ligation high </td><Td> 6uL(HS) or 7.5uL(Act5c) </Td></tr><tr><br />
<td> Total </td><Td> 12uL(HS) or 15uL(Act5c) </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-2-14. Transformation of Ligation products</strong><br />
<br><br />
Ligation products were transformed into E. coli XL-1 blue.<br />
<br />
<br><br><br />
<br />
<h2>September 15th</h2><br />
<br><br />
<strong>1-1-56. Purification of candidate plasmid DNAs</strong><br />
<br><br />
We reproduced The candidate pSB1C3-UAS-LacZ DNA was purified from the three independent colonies, pSB1C3-UAS-EGFP DNA from two independent colonies, pSB1C3-G4L4 DNA from three independent colonies by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
Cut check of the candidate pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA<br />
<br><br />
The candidate pSB1C3-UAS-LacZ DNA from two independent colonies, the candidate pSB1C3-UAS-EGFP DNA from two independent colonies and pSB1C3-G4L4 DNA from three independent colonies were double digested with EcoRI and SpeI. <br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA sample </Td><Td> 1uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 1uL </Td></tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.2uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.2uL </Td></Tr><Tr><br />
<Td> 100×BSA </Td><Td> 0.1uL </Td></Tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 7.5uL </Td></tr><tr><br />
<td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><br><br />
The digested samples were applied to Agarose gel electrophoresis. From the left side lane to the right, two digested candidate pSB1C3-UAS-LacZ DNA, two digested candidate pSB1C3-UAS-EGFP DNA, three digested candidate pSB1C3-G4L4 are shown.<br />
<br><br><br />
Results<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d3/0915kit.png" width="500" height="300"><br />
<br><br><br />
We identified the properly constructed pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA on the gel ! These are Biobrick parts we successfully constructed.<br />
<br><br><br />
<br />
<br />
<h2>September 17th</h2><br />
<BR><br />
<strong>1-1-59 Cut check of the Biobrick part DNAs</strong><br />
<br><br />
The pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA were purified by QIA prep Spin Miniprep Kit. These DNAs were double digested with EcoRI and SpeI again and the digested samples were applied to Agarose gel electrophoresis.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/2/26/0917kit.png" width="500" height="300"><br />
<br><br><br />
Results: The correct size of inserts were detected in the double digested samples further confirming that the pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA are properly constructed.<br />
<br><br><br />
<strong>1-1-60 Submission of the Biobrick parts</strong><br />
<br><br />
The plasmids pSB1C3-UAS-LacZ, pSB1C3-UAS-EGFP, pSB1C3-G4L4 DNA and the previously prepared pSB1C3-UAS were sent out to the iGEM Headquarter via FedEx.<br />
<br><br><br />
<strong>1-1-61 Preparation of DNA for transfection into cultured Drosophila cells</strong><br />
<br><br />
E. coli carrying pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP were grown for 16 hours at 37℃.<br />
<br><br><br />
<br />
<h2>September 18th</h2><br />
<br><br />
<br><br />
<strong>1-1-63</strong><br><br />
pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP DNAs were purified by the QIA prep Spin Miniprep Kit.<br />
<br><br><br />
<br />
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http://2012.igem.org/Team:KIT-Kyoto/Notebook-week3p
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2012-09-26T13:31:21Z
<p>Kazuko: </p>
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<br><br />
</td><br />
<br />
<br />
<td width="800px" height="510px" valign="top"><br />
<div id="MIGI"><br />
<h2>September 7th</h2><br />
<br><br />
<strong>1-1-33 and 2-1-23 PCR amplification of UAS, UAS-TNFAIP3, pSB1C3, GAL4 DNA</strong><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.25uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 34.15uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-34 and 2-1-24 PCR products were applied to the agarose gel electrophoresis</strong><br />
<br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 as shown below<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/0907akit.png" width="500" height="300"><br />
<br><br><br />
Results: no clearly amplified bands were detected.<br />
<br><br />
<br>Re-estimation of the concentration of pSB1C3 and GAL4 DNA used for ligation on 9/5, 6<br />
<br><br><br />
<strong>2-1-25 SB1C3 and GAL4 DNA were applied to the agarose gel electrophoresis</strong><br />
<br><br />
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker - 2uL, DNA 0.1uL, 1kbmarker 1uL<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/68/0907bkit.png" width="500" height="300"><br />
<br><br><br />
From these results, concentration of the pSB1C3 DNA was estimated to be 10ng/uL.<br />
<br><br />
<br><br />
GAL4<br />
<br><br />
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker 2uL, DNA 0.1uL, 1kbmarker 1uL<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/0907ckit.png" width="500" height="300"><br />
<br><br><br />
However no GAL DNA was detected. We lost the sample somewhere by some mistakes.<br />
<br><br><br />
<br />
<br />
<h2>September 8th</h2><br />
<br><br />
<strong>1-1-35 and 2-1-26</strong><br />
<br>PCR amplification of UAS, UAS-TNFAIP3, pSB1C3 and GAL4 DNA<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td></td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-36 and 2-1-27 PCRproducts applied to the agarose gel electrophoresis</strong><br />
<br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 10 uL each<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/be/0908kit.png" width="500" height="300"><br />
<br><br><br />
Only the PCR products for pSB1C3 was detectable.<br />
<br><br />
<br><br />
<strong>1-1-37 and 2-1-28</strong><br />
<br><br />
We repeated PCR for UAS and GAL4DNA by changing the annealing temperature<br />
<br><br />
(-1 indicates 55℃、-2 indicates 58℃)<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 55℃(-1) or 58℃(-2) </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<h2>September 9th</h2><br />
<BR><br />
<strong>1-1-38 and 2-1-29</strong><br />
<br><br />
PCRproducts were electrophoreses.<br />
<br><br />
Left to right: UAS-1, UAS-2, GAL4-1, GAL4-2 10 uL each<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/1/10/0909akit.png" width="500" height="300"><br />
<br><br><br />
We finally succeeded the amplification of the UAS fragments.<br />
<br><br><br />
<strong>1-1-39 Purification of pSB1C3 and UAS-1-PCR products</strong><br />
<br><br />
PCR products were purified by High Pure PCR Product Kit<br />
<br><br><br />
<strong>1-1-40 </strong><br />
<br><br />
The purified UAS fragments were digested with EcoRⅠ and SpeⅠ in the following reaction<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> UAS </Td><Td> 40uL </Td></Tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<Tr><td> SpeⅠ(NEB </td><td> 0.5uL </td></tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 3.5uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-41</strong><br />
<br><br />
The digested UAS DNA was applied to the agarose gel electrophoresis and purified from the gel by QIA quick Gel Extraction Kit<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/91/0909bkit.png" width="500" height="300"><br />
<br><br><br />
<strong>2-1-30 PCR amplification of GAL4</strong><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs <td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min10sec </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 2min10sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-1-31</strong><br />
<br><br />
PCR products were applied to the agarose gel electrophoresis as shown below.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/bb/0909ckit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-1-42</strong><br />
<br><br />
pSB1C3 and UAS fragments were ligated in the following reactions.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (EcoRⅠ and SpeⅠ digested) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> UAS (EcoRⅠ and SpeⅠ digested) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3uL </Td></Tr><br />
<Tr><td> Ligation high </td><td> 2.5uL </td></tr><br />
<Tr><Td> Total </Td><Td> 7.5uL </Td></Tr><br />
</Table><br />
<br><BR><br />
The following reaction were carried out as a negative control. <br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (EcoRⅠ and SpeⅠ digested)</Td><Td> 1uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 4uL </Td></Tr><br />
<Tr><td> Ligation high </td><td> 2.5uL </td></tr><br />
<Tr><Td> Total </Td><Td> 7.5uL </Td></Tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-43</strong><br />
<br><br />
Ligation products(1-1-42) were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(+) plate and incubated at 37℃ for 16 hours.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<h2>September 10th</h2><br />
<br><br />
At 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP-TNFAIP3 DNA was added to the well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 hours at 25 ℃.<br />
<br><br />
<br><br />
<strong>1-1-44 Single colony isolation of ligationproducts</strong><br />
<br><br />
Ligation product prepared on 9/9 (UAS and pSB1C3) gave us some colonies and negative control sample gave us no colony. We therefore picked up four independent colonies and cultured them in 2.5mL LB Chloramphenicol (+) medium at 37℃ for 16 hours.<br />
<br><br><br />
<strong>2-1-32 Digestion of HS promoter fragment and Act5C promoter with XbaⅠ and BamHⅠ</strong><br />
<br><br />
DNA fragments carrying HS promoter (prepared on 9/5) and those carrying Act5C promoter (prepared on 9/3) were digested with XbaⅠ and BamHⅠ.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> HS or Act5c </Td><Td> 40uL </Td></Tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<Tr><td> XbaⅠ-HF(NEB) <td> 0.5uL </td></tr><br />
<Tr><Td> BamHⅠ(NEB )</Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3.5uL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 50uL </Td></Tr><br />
</Table><br />
<br><BR><br />
Digested fragments were purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/27/0910akit.png" width="500" height="300"><br />
<br><br><br />
<strong>2-1-33 PCR amplification of GAL4 fragments</strong><br />
<br><br />
We tried PCR under four different conditions described below. <br />
<Br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><Td> G-1 and G-2 </Td><Td> G-3 and G-4 </Td></Tr><br />
<tr><td> DNA template </td><td> 1uL </td><Td> 4uL </Td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 5uL </td><Td> 5uL </Td></tr><tr><br />
<td> 25mM MgSO<sub>4</sub> </td><td> 1.6uL </td><Td> 1.6uL </Td></tr><br />
<tr><td> 10P 5’ primer </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> 10P 3’ primer </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> KOD plus </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td><Td> 31.4uL </Td></tr><br />
<tr><td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 57℃(G-1 and G-3) or 59℃(G-2 and G-4) </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min10sec </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 2min10sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-1-34 PCR products were applied to agarose gel electrophoresis </strong><br />
<br><br />
From left to right: 10uL each G-1, G-2, G-3, G-4<br />
<br><br />
<br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/eb/0910bkit.png" width="500" height="300"><br />
<br><br><br />
We found that GAL4 sequence was properly amplified under G-1 and G-2conditions.<br />
<br><br><br />
<strong>2-1-35</strong><br />
<br><br />
PCR products were purified by High Pure PCR Product Kit.<br />
<br><br><br />
<strong>1-2-1 Digestion of pSB1C3 DNA with EcoRⅠ and SpeⅠ</strong><br />
<br><br />
PCR-amplified pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ for 2 hours at 37℃.<br />
<br><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3(PCR) </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> SpeⅠ(NEB) </td><Td> 0.5uL </Td></tr><tr><br />
<td> 2100×BSA </td><Td> 0.5uL </Td></tr><br />
<tr><td> dH<sub>2</sub>O <Td> 3.5uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-2-2 Digestion of EcoRI-digested UAS fragment by BglⅡ</strong><br />
<br><br />
BglⅡ-digestion was carried out for 2 hours at 37℃ under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> UAS(EcoRⅠ-digested) </Td><Td> 13uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><Td> 2uL </Td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O <Td> 2.5uL </Td></tr><br />
<tr><td> Total </td><Td> 20uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-2-3 Purification of restriction enzyme-digested pSB1C3 DNA and UAS fragment</strong><br />
<br><br />
restriction enzyme-digested pSB1C3 DNA and UAS fragment (prepared on 9/10) were purified by QIA quick Gel Extraction Kit. <br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/86/0910ckit.png" width="500" height="300"><br />
<br><br><br />
<strong>2-1-36</strong><br />
<br><br />
PCR-amplified GAL4 fragments were digested with XbaⅠ and SpeⅠ under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 20uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 4uL </Td></tr><br />
<Tr><Td> XbaⅠ(NEB) </Td><Td> 0.7uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.7uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.4uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 14.6uL </Td></tr><br />
<tr><td> Total </td><Td> 40uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA was applied to agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/3/39/0910dkit.png" width="500" height="300"><br />
<br><br><br />
Results: XbaⅠ-digestion of GAL4 fragment gave two DNA fragments, indicating that GAL4 sequence contains XbaⅠ site.<br />
<br><br><br />
<strong>2-2-1 Digestion of GAL4 sequence with BglⅡ and SpeⅠ</strong><br />
<br><br />
Digestion was carried out st 37℃ for 1hour under the conditions described below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 4.5uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA was purified by High Pure PCR Product Kit was further digested with Spe I at 37℃ for 1hour.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4(Bgl Ⅱ cut) </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 4uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested fragments were purified by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/0910ekit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-2-4 and 2-2-2</strong><br />
<br><br />
Insertion of GAL4 fragments and HS promoter or Act5C promoter-enhancer and UASfragment and EGFP sequence or LacZ sequence into the pSB1C3DNA<br />
<br><br><br />
Ligation reactions were carried out at 16℃ for 1hour under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCRproducts)(double digested with EcoRⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> UAS (double digested with EcoRⅠ and BglⅡ) </td><Td> 2uL </Td></tr><br />
<Tr><Td> EGFP or LacZ </Td><Td> 2uL </Td></Tr><br />
<tr><td> Ligation high </td><Td> 5uL </Td></tr><br />
<tr><td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (vector DNA) (double digested with XbaⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (double digested with BglⅡ and SpeⅠ) </td><Td> 2uL </Td></tr><br />
<Tr><Td>HS or Act5c (double digested with XbaⅠ and BamHⅠ)</Td><Td> 2uL </Td></Tr><br />
<tr><td> Ligation high </td><Td> 5uL </Td></tr><br />
<tr><td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-2-5 and 2-2-3</strong><br />
<br><br />
Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(+) plate and cultured at 37℃for 16 hours.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 11th</h2><br />
<br><br />
<strong>1-2-6 Single colony isolation </strong><br />
<br><br />
Single colonies were isolated from the plate prepared on 9/11 and cultured in 2.5mL LB Chloramphenicol(+) medium at 37℃ for 16 hours.<br />
<br><br><br />
<strong>1-1-45 Isolation of the candidate pSB1C3-UAS DNA</strong><br />
<br><br />
The candidate pSB1C3-UAS DNA was purified by QIA prep Spin Miniprep Kit.<br />
<br><Br><br />
<strong>1-1-46</strong><br />
<br><br />
The purified candudate pSB1C3-UAS was digested with BglII for 2 hours.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS-1, -2, -3, -4 </Td><Td> 5uL </Td></Tr><br />
<tr><td> 3buffer(NEB) </td><Td> 2uL </Td></tr><br />
<Tr><Td> BglⅡ </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><Td> 12.5uL </Td></tr><br />
<td> Total </td><Td> 20uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were applied to agarose gel electrophoresis as shown below. Left to right: pSB1C3-1 uncut, pSB1C3 cut, -2 uncut, -2 cut, -3 uncut, -3 cut, -4 uncut, -4 cut<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/0/0c/0911akit.png" width="500" height="300"><br />
<br><br><br />
Results: All DNAs examined had a single BglII site, indicating that the pSB1C3-UAS DNA was successfully constructed.<br />
<br><br><br />
The BglII-digested pSB1C3-UAS DNA was purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
<strong>2-2-3 Digestion of pSB1C3 (PCR) with XbaⅠ and SpeⅠ</strong><br />
<br><br />
PCR-amplified pSB1C3 DNA was double digested with XbaⅠ and SpeⅠ at 37℃ for 2 hours. <br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> XbaⅠ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.5uL </Td></Tr><Tr><br />
<Td> CIP </Td><Td> 0.5uL </Td></Tr><Tr><br />
<Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><Td> 3uL </Td></tr><br />
<td> Total </td><Td> 40uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were purified by QIA quick Gel Extraction Kit<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/da/0911kit.png" width="500" height="300"><br />
<br><br><br />
<strong>2-2-4 Insertion of GAL4, HS promoter or Act5C promoter-enhancer into the pSB1C3DNA</strong><br />
<br><br />
Ligation reactions were carried out under conditions described below at 16℃ for 1hour .<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (vector) (digested with XbaⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (digestion with BglⅡ and SpeⅠ) </td><Td> 2.5uL </Td></tr><br />
<Tr><Td> HS promoter or Act5C promoter-enhancer (digested with XbaⅠ and BamHⅠ) </Td><Td> 2.5uL </Td></Tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCR products) (digested with XbaⅠ and SpeⅠ) </Td><Td> 2uL </Td></Tr><br />
<tr><td> GAL4 (digested with BglⅡ and SpeⅠ) </td><Td> 2.5uL </Td></tr><br />
<Tr><Td> HS promoter or Act5C promoter-enhancer (XbaⅠ and BamHⅠ) </Td><Td> 1.5uL </Td></Tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-2-5</strong><br />
<br><br />
Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(+) plate and cultured at 37℃for 16 hours.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<h2>September 12th</h2><br />
<br><br />
<strong>1-2-7 Purification of the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs</strong><br />
<br><br />
We purified the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs by QIA prep Spin Miniprep Kit from E. coli cultured in LB medium for 16 hours (9/11). <br />
<br><br><br />
<strong>1-1-47 Digestion of pSB1C3-UAS DNA by Spe1</strong><br />
<br><br />
We incubated BglⅡ-digested pSB1C3-UAS (9/11) DNA with SpeⅠfor 2 hours. The reaction conditions are shown below.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS-1, -2 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 1uL </Td></Tr><br />
<td> 100×BSA </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 3.5uL </Td></tr><br />
<td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA were applied to agarose gel electrophoresis. We isolated DNA from the gel by using QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of Agarose gel.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/a/ab/0912kit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-1-48 and 2-2-6 Ligation</strong><br />
<br><br />
DNA fragment carrying EGFP or LacZ was ligated into the BglII and SpeI digested pSB1C3-UAS DNA .for 1hour at 16℃.<br />
<br><br><br />
Ligation reactions are listed below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS (double digested with BglII and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> EGFP fragments or LacZ fragments </td><Td> 2uL </Td></tr><br />
<Tr><Td> EGFP or LacZ </Td><Td> 2uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 2.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 2uL </Td></tr><tr><br />
<td> Total </td><Td> 7.5uL </Td></tr><br />
</Table><br />
<br><BR><br />
We ligated DNA fragment carrying GAL4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA for 1hour at 16℃.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCR) (double digested with XbaⅠand SpeI) </Td><Td> 2uL </Td></Tr><br />
<tr><td> GAL4(double digested with BglⅡ and SpeⅠ) </td><Td> 2uL </Td></tr><br />
<Tr><Td> HS fragments or Act5c fragments (double digested with XbaⅠand BamHⅠ) </Td><Td> 2uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-49 and 2-2-7 Transformation of E. coli by Ligation products</strong><br />
<br><br />
We did transformation of E. coli Xl-1 blue with each of ligation products. Finally, we spread them on the LB Chloramphenicol(+) plate and incubated for 16 hours at 37℃.<br />
<br />
<br><br><br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week4p">>>>>>>>>>WEEK4</a></div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week3p
Team:KIT-Kyoto/Notebook-week3p
2012-09-26T07:00:31Z
<p>Kazuko: </p>
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<h2>September 7th</h2><br />
<br><br />
<strong>1-1-33 and 2-1-23 PCR amplification of UAS, UAS-TNFAIP3, pSB1C3, GAL4 DNA</strong><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.25uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 34.15uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-34 and 2-1-24 PCR products were applied to the agarose gel electrophoresis</strong><br />
<br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 as shown below<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/0907akit.png" width="500" height="300"><br />
<br><br><br />
Results: no clearly amplified bands were detected.<br />
<br><br />
<br>Re-estimation of the concentration of pSB1C3 and GAL4 DNA used for ligation on 9/5, 6<br />
<br><br><br />
<strong>2-1-25 SB1C3 and GAL4 DNA were applied to the agarose gel electrophoresis</strong><br />
<br><br />
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker - 2uL, DNA 0.1uL, 1kbmarker 1uL<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/68/0907bkit.png" width="500" height="300"><br />
<br><br><br />
From these results, concentration of the pSB1C3 DNA was estimated to be 10ng/uL.<br />
<br><br />
<br><br />
GAL4<br />
<br><br />
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker 2uL, DNA 0.1uL, 1kbmarker 1uL<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/0907ckit.png" width="500" height="300"><br />
<br><br><br />
However no GAL DNA was detected. We lost the sample somewhere by some mistakes.<br />
<br><br><br />
<br />
<br />
<h2>September 8th</h2><br />
<br><br />
<strong>1-1-35 and 2-1-26</strong><br />
<br>PCR amplification of UAS, UAS-TNFAIP3, pSB1C3 and GAL4 DNA<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td></td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-36 and 2-1-27 PCRproducts applied to the agarose gel electrophoresis</strong><br />
<br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 10 uL each<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/be/0908kit.png" width="500" height="300"><br />
<br><br><br />
Only the PCR products for pSB1C3 was detectable.<br />
<br><br />
<br><br />
<strong>1-1-37 and 2-1-28</strong><br />
<br><br />
We repeated PCR for UAS and GAL4DNA by changing the annealing temperature<br />
<br><br />
(-1 indicates 55℃、-2 indicates 58℃)<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 55℃(-1) or 58℃(-2) </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<h2>September 9th</h2><br />
<BR><br />
<strong>1-1-38 and 2-1-29</strong><br />
<br><br />
PCRproducts were electrophoreses.<br />
<br><br />
Left to right: UAS-1, UAS-2, GAL4-1, GAL4-2 10 uL each<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/1/10/0909akit.png" width="500" height="300"><br />
<br><br><br />
We finally succeeded the amplification of the UAS fragments.<br />
<br><br><br />
<strong>1-1-39 Purification of pSB1C3 and UAS-1-PCR products</strong><br />
<br><br />
PCR products were purified by High Pure PCR Product Kit<br />
<br><br><br />
<strong>1-1-40 </strong><br />
<br><br />
The purified UAS fragments were digested with EcoRⅠ and SpeⅠ in the following reaction<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> UAS </Td><Td> 40uL </Td></Tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<Tr><td> SpeⅠ(NEB </td><td> 0.5uL </td></tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 3.5uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-41</strong><br />
<br><br />
The digested UAS DNA was applied to the agarose gel electrophoresis and purified from the gel by QIA quick Gel Extraction Kit<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/91/0909bkit.png" width="500" height="300"><br />
<br><br><br />
<strong>2-1-30 PCR amplification of GAL4</strong><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs <td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min10sec </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 2min10sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-1-31</strong><br />
<br><br />
PCR products were applied to the agarose gel electrophoresis as shown below.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/bb/0909ckit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-1-42</strong><br />
<br><br />
pSB1C3 and UAS fragments were ligated in the following reactions.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (EcoRⅠ and SpeⅠ digested) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> UAS (EcoRⅠ and SpeⅠ digested) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3uL </Td></Tr><br />
<Tr><td> Ligation high </td><td> 2.5uL </td></tr><br />
<Tr><Td> Total </Td><Td> 7.5uL </Td></Tr><br />
</Table><br />
<br><BR><br />
The following reaction were carried out as a negative control. <br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (EcoRⅠ and SpeⅠ digested)</Td><Td> 1uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 4uL </Td></Tr><br />
<Tr><td> Ligation high </td><td> 2.5uL </td></tr><br />
<Tr><Td> Total </Td><Td> 7.5uL </Td></Tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-43</strong><br />
<br><br />
Ligation products(1-1-42) were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(+) plate and incubated at 37℃ for 16 hours.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<h2>September 10th</h2><br />
<br><br />
At 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP-TNFAIP3 DNA was added to the well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 hours at 25 ℃.<br />
<br><br />
<br><br />
<strong>1-1-44 Single colony isolation of ligationproducts</strong><br />
<br><br />
Ligation product prepared on 9/9 (UAS and pSB1C3) gave us some colonies and negative control sample gave us no colony. We therefore picked up four independent colonies and cultured them in 2.5mL LB Chloramphenicol (+) medium at 37℃ for 16 hours.<br />
<br><br><br />
<strong>2-1-32 Digestion of HS promoter fragment and Act5C promoter with XbaⅠ and BamHⅠ</strong><br />
<br><br />
DNA fragments carrying HS promoter (prepared on 9/5) and those carrying Act5C promoter (prepared on 9/3) were digested with XbaⅠ and BamHⅠ.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> HS or Act5c </Td><Td> 40uL </Td></Tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<Tr><td> XbaⅠ-HF(NEB) <td> 0.5uL </td></tr><br />
<Tr><Td> BamHⅠ(NEB )</Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3.5uL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 50uL </Td></Tr><br />
</Table><br />
<br><BR><br />
Digested fragments were purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/27/0910akit.png" width="500" height="300"><br />
<br><br><br />
<strong>2-1-33 PCR amplification of GAL4 fragments</strong><br />
<br><br />
We tried PCR under four different conditions described below. <br />
<Br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><Td> G-1 and G-2 </Td><Td> G-3 and G-4 </Td></Tr><br />
<tr><td> DNA template </td><td> 1uL </td><Td> 4uL </Td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 5uL </td><Td> 5uL </Td></tr><tr><br />
<td> 25mM MgSO<sub>4</sub> </td><td> 1.6uL </td><Td> 1.6uL </Td></tr><br />
<tr><td> 10P 5’ primer </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> 10P 3’ primer </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> KOD plus </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td><Td> 31.4uL </Td></tr><br />
<tr><td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 57℃(G-1 and G-3) or 59℃(G-2 and G-4) </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min10sec </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 2min10sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-1-34 PCR products were applied to agarose gel electrophoresis </strong><br />
<br><br />
From left to right: 10uL each G-1, G-2, G-3, G-4<br />
<br><br />
<br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/eb/0910bkit.png" width="500" height="300"><br />
<br><br><br />
We found that GAL4 sequence was properly amplified under G-1 and G-2conditions.<br />
<br><br><br />
<strong>2-1-35</strong><br />
<br><br />
PCR products were purified by High Pure PCR Product Kit.<br />
<br><br><br />
<strong>1-2-1 Digestion of pSB1C3 DNA with EcoRⅠ and SpeⅠ</strong><br />
<br><br />
PCR-amplified pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ for 2 hours at 37℃.<br />
<br><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3(PCR) </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> SpeⅠ(NEB) </td><Td> 0.5uL </Td></tr><tr><br />
<td> 2100×BSA </td><Td> 0.5uL </Td></tr><br />
<tr><td> dH<sub>2</sub>O <Td> 3.5uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-2-2 Digestion of EcoRI-digested UAS fragment by BglⅡ</strong><br />
<br><br />
BglⅡ-digestion was carried out for 2 hours at 37℃ under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> UAS(EcoRⅠ-digested) </Td><Td> 13uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><Td> 2uL </Td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O <Td> 2.5uL </Td></tr><br />
<tr><td> Total </td><Td> 20uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-2-3 Purification of restriction enzyme-digested pSB1C3 DNA and UAS fragment</strong><br />
<br><br />
restriction enzyme-digested pSB1C3 DNA and UAS fragment (prepared on 9/10) were purified by QIA quick Gel Extraction Kit. <br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/86/0910ckit.png" width="500" height="300"><br />
<br><br><br />
<strong>2-1-36 PCR-amplified GAL4 fragments were digested with XbaⅠ and SpeⅠ under conditions described below.</strong><br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 20uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 4uL </Td></tr><br />
<Tr><Td> XbaⅠ(NEB) </Td><Td> 0.7uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.7uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.4uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 14.6uL </Td></tr><br />
<tr><td> Total </td><Td> 40uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA was applied to agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/3/39/0910dkit.png" width="500" height="300"><br />
<br><br><br />
Results: XbaⅠ-digestion of GAL4 fragment gave two DNA fragments, indicating that GAL4 sequence contains XbaⅠ site.<br />
<br><br><br />
<strong>2-2-1 Digestion of GAL4 sequence with BglⅡ and SpeⅠ</strong><br />
<br><br />
Digestion was carried out st 37℃ for 1hour under the conditions described below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 4.5uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA was purified by High Pure PCR Product Kit was further digested with Spe I at 37℃ for 1hour.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4(Bgl Ⅱ cut) </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 4uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested fragments were purified by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/0910ekit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-2-4 and 2-2-2 Insertion of GAL4 fragments and HS promoter or Act5C promoter-enhancer and UASfragment and EGFP sequence or LacZ sequence into the pSB1C3DNA</strong><br />
<br><br><br />
Ligation reactions were carried out at 16℃ for 1hour under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCRproducts)(double digested with EcoRⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> UAS (double digested with EcoRⅠ and BglⅡ) </td><Td> 2uL </Td></tr><br />
<Tr><Td> EGFP or LacZ </Td><Td> 2uL </Td></Tr><br />
<tr><td> Ligation high </td><Td> 5uL </Td></tr><br />
<tr><td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (vector DNA) (double digested with XbaⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (double digested with BglⅡ and SpeⅠ) </td><Td> 2uL </Td></tr><br />
<Tr><Td>HS or Act5c (double digested with XbaⅠ and BamHⅠ)</Td><Td> 2uL </Td></Tr><br />
<tr><td> Ligation high </td><Td> 5uL </Td></tr><br />
<tr><td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-2-5 and 2-2-3</strong><br />
<br><br />
Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(+) plate and cultured at 37℃for 16 hours.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 11th</h2><br />
<br><br />
<strong>1-2-6 Single colony isolation </strong><br />
<br><br />
Single colonies were isolated from the plate prepared on 9/11 and cultured in 2.5mL LB Chloramphenicol(+) medium at 37℃ for 16 hours.<br />
<br><br><br />
<strong>1-1-45 Isolation of the candidate pSB1C3-UAS DNA</strong><br />
<br><br />
The candidate pSB1C3-UAS DNA was purified by QIA prep Spin Miniprep Kit.<br />
<br><Br><br />
<strong>1-1-46</strong><br />
<br><br />
The purified candudate pSB1C3-UAS was digested with BglII for 2 hours.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS-1, -2, -3, -4 </Td><Td> 5uL </Td></Tr><br />
<tr><td> 3buffer(NEB) </td><Td> 2uL </Td></tr><br />
<Tr><Td> BglⅡ </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><Td> 12.5uL </Td></tr><br />
<td> Total </td><Td> 20uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were applied to agarose gel electrophoresis as shown below. Left to right: pSB1C3-1 uncut, pSB1C3 cut, -2 uncut, -2 cut, -3 uncut, -3 cut, -4 uncut, -4 cut<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/0/0c/0911akit.png" width="500" height="300"><br />
<br><br><br />
Results: All DNAs examined had a single BglII site, indicating that the pSB1C3-UAS DNA was successfully constructed.<br />
<br><br><br />
The BglII-digested pSB1C3-UAS DNA was purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
<strong>2-2-3 Digestion of pSB1C3 (PCR) with XbaⅠ and SpeⅠ</strong><br />
<br><br />
PCR-amplified pSB1C3 DNA was double digested with XbaⅠ and SpeⅠ at 37℃ for 2 hours. <br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> XbaⅠ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.5uL </Td></Tr><Tr><br />
<Td> CIP </Td><Td> 0.5uL </Td></Tr><Tr><br />
<Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><Td> 3uL </Td></tr><br />
<td> Total </td><Td> 40uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were purified by QIA quick Gel Extraction Kit<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/da/0911kit.png" width="500" height="300"><br />
<br><br><br />
<strong>2-2-4 Insertion of GAL4, HS promoter or Act5C promoter-enhancer into the pSB1C3DNA</strong><br />
<br><br />
Ligation reactions were carried out under conditions described below at 16℃ for 1hour .<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (vector) (digested with XbaⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (digestion with BglⅡ and SpeⅠ) </td><Td> 2.5uL </Td></tr><br />
<Tr><Td> HS promoter or Act5C promoter-enhancer (digested with XbaⅠ and BamHⅠ) </Td><Td> 2.5uL </Td></Tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCR products) (digested with XbaⅠ and SpeⅠ) </Td><Td> 2uL </Td></Tr><br />
<tr><td> GAL4 (digested with BglⅡ and SpeⅠ) </td><Td> 2.5uL </Td></tr><br />
<Tr><Td> HS promoter or Act5C promoter-enhancer (XbaⅠ and BamHⅠ) </Td><Td> 1.5uL </Td></Tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-2-5</strong><br />
<br><br />
Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(+) plate and cultured at 37℃for 16 hours.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<h2>September 12th</h2><br />
<br><br />
<strong>1-2-7 Purification of the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs</strong><br />
<br><br />
We purified the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs by QIA prep Spin Miniprep Kit from E. coli cultured in LB medium for 16 hours (9/11). <br />
<br><br><br />
<strong>1-1-47 Digestion of pSB1C3-UAS DNA by Spe1</strong><br />
<br><br />
We incubated BglⅡ-digested pSB1C3-UAS (9/11) DNA with SpeⅠfor 2 hours. The reaction conditions are shown below.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS-1, -2 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 1uL </Td></Tr><br />
<td> 100×BSA </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 3.5uL </Td></tr><br />
<td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA were applied to agarose gel electrophoresis. We isolated DNA from the gel by using QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of Agarose gel.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/a/ab/0912kit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-1-48 and 2-2-6 Ligation</strong><br />
<br><br />
DNA fragment carrying EGFP or LacZ was ligated into the BglII and SpeI digested pSB1C3-UAS DNA .for 1hour at 16℃.<br />
<br><br><br />
Ligation reactions are listed below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS (double digested with BglII and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> EGFP fragments or LacZ fragments </td><Td> 2uL </Td></tr><br />
<Tr><Td> EGFP or LacZ </Td><Td> 2uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 2.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 2uL </Td></tr><tr><br />
<td> Total </td><Td> 7.5uL </Td></tr><br />
</Table><br />
<br><BR><br />
We ligated DNA fragment carrying GAL4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA for 1hour at 16℃.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCR) (double digested with XbaⅠand SpeI) </Td><Td> 2uL </Td></Tr><br />
<tr><td> GAL4(double digested with BglⅡ and SpeⅠ) </td><Td> 2uL </Td></tr><br />
<Tr><Td> HS fragments or Act5c fragments (double digested with XbaⅠand BamHⅠ) </Td><Td> 2uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-49 and 2-2-7 Transformation of E. coli by Ligation products</strong><br />
<br><br />
We did transformation of E. coli Xl-1 blue with each of ligation products. Finally, we spread them on the LB Chloramphenicol(+) plate and incubated for 16 hours at 37℃.<br />
<br />
<br><br><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week2p
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2012-09-26T06:44:51Z
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<h2>August 31st</h2><br />
<br><br />
<strong>1-1-16 and 2-1-6 <br />
PCR amplification of DNA fragments containing UAS and Heat Shock promoter</strong><br />
<br><br />
We have carried out the PCR reaction for UAS again. PCR amplification of Heat Shock promoter from pCasPer DNA was also carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 10uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 10uL </Td></Tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 3.2uL </Td></Tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<Tr><td> KOD plus </td><td> 2uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<Tr><td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature</Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<strong> 2-1-7 Purification of pAct5C DNA and pGaTB DNA</strong><br><br />
pAct5C DNA and pGaTB DNA was purified from E. coli prepared on 8/29by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
<br />
<h2>September 1st</h2><br />
<br><br />
<strong>1-1-18 and 2-1-8 <br />
Agarose gel electrophoresis of PCR produsts and the purified pAct5C DNA and pGaTB DNA</strong><br><br />
<br />
Agarose gel image is shown below. Left to right: HS promoter 10uL, UAS fragment 10uL, pAct5C DNA-1, pGaTB DNA-1, pGaTB DNA-2, pAct5C DNA<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/b/b0/0901akit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-1-19 and 2-1-9 Expected bands for pAct5C DNA and pGaTB DNA were detected on the gel, but band for UAS fragment was not.</strong><br />
<br><br><br />
<br />
<br />
<br />
<h2>September 3rd</h2><br />
<BR><br />
<strong>1-1-21 and 2-1-11<br />
PCR amplification of DNA fragments containing GAL4, Act5C promoter and LacZ sequence</strong><br><br />
pGaTBDNA was used as a template to amplify GAL4 sequence. pAct5C-LacZ DNA was used to amplify Act5C promoter-enhancer region and LacZ. <br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA template </td><td> 0.2uL </td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 5uL </td></tr><tr><br />
<td> 25mM MgSO<sub>4</sub> </td><td> 1.6uL </td></tr><tr><br />
<td> 10P 5’ primer </td><td> 1.5uL </td></tr><tr><br />
<td> 10P 3’ primer </td><td> 1.5uL </td></tr><tr><br />
<td> KOD plus </td><td> 1u L</td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 34.2uL </td></tr><tr><br />
<td> Total <td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Circle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 55℃(GAL4) and 58℃(Except for GAL4) </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min30sec </td><Td> 25 cycle </Td></tr><br />
<td> 68℃ </td><td> 2min30sec </td><Td></Td></tr><br />
<td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><br><br />
<strong>1-1-22 and 2-1-12<br />
Agarose gel electrophoresis of the PCRproducts</strong><br><br />
Left to right: 1kb ladder marker 5uL GAL4 fragments 10uL Act5C promoter-enhancer 10uL LacZ sequence <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/c/ca/0903kit.png" width="500" height="300"><br />
<br><br><br />
Results: Expected PCR products were all detected on the gel.<br />
<br><br />
<br><br />
<strong>1-1-23 and 2-1-13</strong><br><br />
<br><br />
These PCR products were purified by High Pure PCR Product Kit (Roche).<br />
<br><br><br />
<br />
<h2>September 4th</h2><br />
<br><br />
<strong>2-1-14<br />
<br />
Digestion of GAL4 fragments by XbaⅠ and SpeⅠ</strong><br><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 fragments prepared on 9/3 </Td><Td> 40uL </Td></Tr><br />
<tr><td> M buffer(TOYOBO) </td><td> 5uL </td></tr><br />
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> SpeⅠ(TOYOBO) </td><td> 0.5uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 4uL </td></tr><tr><br />
<td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-24<br />
Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ</strong><br><br />
pSB1C3 DNA prepared on 8/27, LacZ fragment prepared on 9/3 and EGFP fragments were digested with BglⅡ in the following conditions.<br />
<br><br />
<br><br />
<strong>1-1-25 Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ</strong><br />
<br><br />
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> each DNA </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><td> 5uL </td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 4.5uL </td></tr><tr><br />
<td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
<br />
<strong>2-1-15</strong><br><br />
Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ<br><br />
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.<br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 DNA </Td><Td> 23uL </Td></Tr><br />
<tr><td> M buffer(TOYOBO) </td><td> 10uL </td></tr><br />
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> SpeⅠ(TOYOBO) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> CIP </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><td> 64.5uL </td></tr><br />
<td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-1-16<br />
Purification of GAL4 fragments</strong><br><br />
After agarose gel electrophoresis, the digested GAL4 fragments were purified by QIA quick Gel Extraction Kit<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/b0/0904kit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-1-25<br />
Purification of BglII-digested LacZ fragments</strong><br><br />
BglII-digested LacZ fragments were purified by High Pure PCR Product Kit.<br />
<br><br><br />
<br />
<h2>September 5th</h2><br />
<br><br />
<strong>1-1-26 and 2-1-17<br />
Purification of EGFP fragments and HS promoter fragments</strong><br><br />
EGFP fragments prepared on 8/29 and HS promoter fragments prepared on 8/31were purified by High Pure PCR Product Kit<br />
<br><br><br />
<strong>1-1-27<br />
EGFP fragments and pSB1C3 DNA prepared on 8/27were digested with BglⅡ in the following reaction.</strong><br><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><td> EGFP </td><Td> pSB1C3 </Td></Tr><br />
<tr><td> DNA template </td><td> 40uL </td><Td> 23uL </Td></tr><br />
<Tr><Td> 3 buffer </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> BglⅡ </td><td> 0.5uL </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 4.5uL </td><Td> 21.5uL </Td></tr><br />
<td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-1-18<br />
<br />
Purification of pSB1C3DNA digested with XbaⅠ and SpeⅠ<br />
</strong><br><br />
Agarose gel electrophoresis image of the purified sample are shown below.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/3/3a/0905akit.png" width="500" height="300"><br />
<br><br><br />
<br />
<strong>1-1-28<br />
<br />
SpeI digestion of the BglⅡ-digested LacZ, EGFP, pSB1C3 DNA</strong><br><br />
SpeⅠ digestion was carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><td> 5uL </td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> CIP </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><td> 4uL </td></tr><br />
<td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-1-19<br />
<br />
Ligation of pSB1C3 DNA and GAL4 fragment</strong><br><br />
Ligation was carried out in the following reactions.<br />
<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (XbaⅠand SpeⅠdigested) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (XbaⅠ and SpeⅠ digested) </td><td> 1uL </td></tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3uL </Td></Tr><br />
<Tr><Td> Ligation high </Td><Td> 2.5uL </Td></Tr><br />
<td> Total </td><td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-1-20</strong><br><br />
<br />
The ligated products were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(+) plate<br />
<br><br><br />
<strong>1-1-29<br />
<br />
Purification of pSB1C3, EGFP, LacZfragments double digested with BglⅡ and SpeⅠ<br />
</strong><br><br />
The double digested samples were applied to the agarose gel electrophoresis and the DNA fragments were purified by QIA quick Gel Extraction Kit<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/63/0905bkit.png" width="500" height="300"><br />
<br><br><br />
<br />
<br />
<h2>September 6th</h2><br />
<br><br />
<br><br />
<br />
Transformation(2-1-20) performed on September 5th was not successful, since we had no colony on the plate. <br />
<br><br><br />
<br />
<br />
<strong>1-1-30</strong><br><br />
<br />
PCR amplification of UAS , UAS-TNFAIP3, pSB1C3DNA<br />
<br><br />
<br><br />
Composition<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.25uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 5uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 5uL </Td></Tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><tr><br />
<td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 34.15uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
<br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec</Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec</td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<strong>1-1-31 PCR products were applied to the agarose gel electrophoresis</strong><br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3<br />
<br><br><br />
<br />
<strong>1-1-32 Purifucation of pSB1C3 PCR products</strong><br><br />
<br />
pSB1C3 PCR products were purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/5/53/0906kit.png" width="500" height="300"><br />
<br><br><br />
<strong> 2-1-21 Ligation of pSB1C3 DNA and GAL4 fragment</strong><br><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3(XbaⅠ and SpeⅠ cut) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> GAL4(XbaⅠ and SpeⅠ cut) </Td><Td> 1uL </Td></Tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 3uL </td></tr><br />
<Tr><Td> Ligation high </Td><Td> 2.5uL </Td></Tr><br />
<Tr><td> Total </td><td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-1-22</strong><br><br />
The ligation products were transformed into E. coli XL-1and spread on the LB Chloramphenicol(+) plate<br />
<br><br><br />
<br />
<br />
<br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week3p">>>>>>>>>>WEEK3</a></div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week3p
Team:KIT-Kyoto/Notebook-week3p
2012-09-26T06:40:15Z
<p>Kazuko: </p>
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<div id="MIGI"><br />
<h2>September 7th</h2><br />
<br><br />
<strong>1-1-33 and 2-1-23 PCR amplification of UAS, UAS-TNFAIP3, pSB1C3, GAL4 DNA</strong><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.25uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 34.15uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-34 and 2-1-24 PCR products were applied to the agarose gel electrophoresis</strong><br />
<br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 as shown below<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/0907akit.png" width="500" height="300"><br />
<br><br><br />
Results: no clearly amplified bands were detected.<br />
<br><br />
<br>Re-estimation of the concentration of pSB1C3 and GAL4 DNA used for ligation on 9/5, 6<br />
<br><br><br />
<strong>2-1-25 SB1C3 and GAL4 DNA were applied to the agarose gel electrophoresis</strong><br />
<br><br />
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker - 2uL, DNA 0.1uL, 1kbmarker 1uL<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/68/0907bkit.png" width="500" height="300"><br />
<br><br><br />
From these results, concentration of the pSB1C3 DNA was estimated to be 10ng/uL.<br />
<br><br />
<br><br />
GAL4<br />
<br><br />
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker 2uL, DNA 0.1uL, 1kbmarker 1uL<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/0907ckit.png" width="500" height="300"><br />
<br><br><br />
However no GAL DNA was detected. We lost the sample somewhere by some mistakes.<br />
<br><br><br />
<br />
<br />
<h2>September 8th</h2><br />
<br><br />
<strong>1-1-35 and 2-1-26</strong><br />
<br>PCR amplification of UAS, UAS-TNFAIP3, pSB1C3 and GAL4 DNA<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td></td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-36 and 2-1-27 PCRproducts applied to the agarose gel electrophoresis</strong><br />
<br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 10 uL each<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/be/0908kit.png" width="500" height="300"><br />
<br><br><br />
Only the PCR products for pSB1C3 was detectable.<br />
<br><br />
<br><br />
<strong>1-1-37 and 2-1-28</strong><br />
<br><br />
We repeated PCR for UAS and GAL4DNA by changing the annealing temperature<br />
<br><br />
(-1 indicates 55℃、-2 indicates 58℃)<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 55℃(-1) or 58℃(-2) </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<h2>September 9th</h2><br />
<BR><br />
<strong>1-1-38 and 2-1-29</strong><br />
<br><br />
PCRproducts were electrophoreses.<br />
<br><br />
Left to right: UAS-1, UAS-2, GAL4-1, GAL4-2 10 uL each<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/1/10/0909akit.png" width="500" height="300"><br />
<br><br><br />
We finally succeeded the amplification of the UAS fragments.<br />
<br><br><br />
<strong>1-1-39 Purification of pSB1C3 and UAS-1-PCR products</strong><br />
<br><br />
PCR products were purified by High Pure PCR Product Kit<br />
<br><br><br />
<strong>1-1-40 </strong><br />
<br><br />
The purified UAS fragments were digested with EcoRⅠ and SpeⅠ in the following reaction<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> UAS </Td><Td> 40uL </Td></Tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<Tr><td> SpeⅠ(NEB </td><td> 0.5uL </td></tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 3.5uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-41</strong><br />
<br><br />
The digested UAS DNA was applied to the agarose gel electrophoresis and purified from the gel by QIA quick Gel Extraction Kit<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/91/0909bkit.png" width="500" height="300"><br />
<br><br><br />
<strong>2-1-30 PCR amplification of GAL4</strong><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs <td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min10sec </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 2min10sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-1-31</strong><br />
<br><br />
PCR products were applied to the agarose gel electrophoresis as shown below.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/bb/0909ckit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-1-42</strong><br />
<br><br />
pSB1C3 and UAS fragments were ligated in the following reactions.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (EcoRⅠ and SpeⅠ digested) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> UAS (EcoRⅠ and SpeⅠ digested) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3uL </Td></Tr><br />
<Tr><td> Ligation high </td><td> 2.5uL </td></tr><br />
<Tr><Td> Total </Td><Td> 7.5uL </Td></Tr><br />
</Table><br />
<br><BR><br />
The following reaction were carried out as a negative control. <br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (EcoRⅠ and SpeⅠ digested)</Td><Td> 1uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 4uL </Td></Tr><br />
<Tr><td> Ligation high </td><td> 2.5uL </td></tr><br />
<Tr><Td> Total </Td><Td> 7.5uL </Td></Tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-43</strong><br />
<br><br />
Ligation products(1-1-42) were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(+) plate and incubated at 37℃ for 16 hours.<br />
<br><br><br />
<br />
<h2>September 10th</h2><br />
<br><br />
At 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP-TNFAIP3 DNA was added to the well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 hours at 25 ℃.<br />
<br><br />
<br><br />
1. Single colony isolation of ligationproducts<br />
<br><br />
Ligation product prepared on 9/9 (UAS and pSB1C3) gave us some colonies and negative control sample gave us no colony. We therefore picked up four independent colonies and cultured them in 2.5mL LB Chloramphenicol (+) medium at 37℃ for 16 hours.<br />
<br><br><br />
2. Digestion of HS promoter fragment and Act5C promoter with XbaⅠ and BamHⅠ<br />
<br><br />
DNA fragments carrying HS promoter (prepared on 9/5) and those carrying Act5C promoter (prepared on 9/3) were digested with XbaⅠ and BamHⅠ.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> HS or Act5c </Td><Td> 40uL </Td></Tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<Tr><td> XbaⅠ-HF(NEB) <td> 0.5uL </td></tr><br />
<Tr><Td> BamHⅠ(NEB )</Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3.5uL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 50uL </Td></Tr><br />
</Table><br />
<br><BR><br />
Digested fragments were purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/27/0910akit.png" width="500" height="300"><br />
<br><br><br />
3. PCR amplification of GAL4 fragments<br />
<br><br />
We tried PCR under four different conditions described below. <br />
<Br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><Td> G-1 and G-2 </Td><Td> G-3 and G-4 </Td></Tr><br />
<tr><td> DNA template </td><td> 1uL </td><Td> 4uL </Td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 5uL </td><Td> 5uL </Td></tr><tr><br />
<td> 25mM MgSO<sub>4</sub> </td><td> 1.6uL </td><Td> 1.6uL </Td></tr><br />
<tr><td> 10P 5’ primer </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> 10P 3’ primer </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> KOD plus </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td><Td> 31.4uL </Td></tr><br />
<tr><td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 57℃(G-1 and G-3) or 59℃(G-2 and G-4) </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min10sec </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 2min10sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
4. PCR products were applied to agarose gel electrophoresis <br />
<br><br />
From left to right: 10uL each G-1, G-2, G-3, G-4<br />
<br><br />
<br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/eb/0910bkit.png" width="500" height="300"><br />
<br><br><br />
We found that GAL4 sequence was properly amplified under G-1 and G-2conditions.<br />
<brr><br />
5. PCR products were purified by High Pure PCR Product Kit.<br />
<br><br />
6. Digestion of pSB1C3 DNA with EcoRⅠ and SpeⅠ<br />
<br><br />
PCR-amplified pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ for 2 hours at 37℃.<br />
<br><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3(PCR) </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> SpeⅠ(NEB) </td><Td> 0.5uL </Td></tr><tr><br />
<td> 2100×BSA </td><Td> 0.5uL </Td></tr><br />
<tr><td> dH<sub>2</sub>O <Td> 3.5uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
7. Digestion of EcoRI-digested UAS fragment by BglⅡ<br />
<br><br />
BglⅡ-digestion was carried out for 2 hours at 37℃ under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> UAS(EcoRⅠ-digested) </Td><Td> 13uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><Td> 2uL </Td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O <Td> 2.5uL </Td></tr><br />
<tr><td> Total </td><Td> 20uL </Td></tr><br />
</Table><br />
<br><BR><br />
8. Purification of restriction enzyme-digested pSB1C3 DNA and UAS fragment<br />
<br><br />
restriction enzyme-digested pSB1C3 DNA and UAS fragment (prepared on 9/10) were purified by QIA quick Gel Extraction Kit. <br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/86/0910ckit.png" width="500" height="300"><br />
<br><br><br />
9. PCR-amplified GAL4 fragments were digested with XbaⅠ and SpeⅠ under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 20uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 4uL </Td></tr><br />
<Tr><Td> XbaⅠ(NEB) </Td><Td> 0.7uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.7uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.4uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 14.6uL </Td></tr><br />
<tr><td> Total </td><Td> 40uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA was applied to agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/3/39/0910dkit.png" width="500" height="300"><br />
<br><br><br />
Results: XbaⅠ-digestion of GAL4 fragment gave two DNA fragments, indicating that GAL4 sequence contains XbaⅠ site.<br />
<br><br><br />
10. Digestion of GAL4 sequence with BglⅡ and SpeⅠ<br />
<br><br />
Digestion was carried out st 37℃ for 1hour under the conditions described below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 4.5uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA was purified by High Pure PCR Product Kit was further digested with Spe I at 37℃ for 1hour.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4(Bgl Ⅱ cut) </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 4uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested fragments were purified by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/0910ekit.png" width="500" height="300"><br />
<br><br><br />
11. Insertion of GAL4 fragments and HS promoter or Act5C promoter-enhancer and UASfragment and EGFP sequence or LacZ sequence into the pSB1C3DNA<br />
<br><br><br />
Ligation reactions were carried out at 16℃ for 1hour under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCRproducts)(double digested with EcoRⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> UAS (double digested with EcoRⅠ and BglⅡ) </td><Td> 2uL </Td></tr><br />
<Tr><Td> EGFP or LacZ </Td><Td> 2uL </Td></Tr><br />
<tr><td> Ligation high </td><Td> 5uL </Td></tr><br />
<tr><td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (vector DNA) (double digested with XbaⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (double digested with BglⅡ and SpeⅠ) </td><Td> 2uL </Td></tr><br />
<Tr><Td>HS or Act5c (double digested with XbaⅠ and BamHⅠ)</Td><Td> 2uL </Td></Tr><br />
<tr><td> Ligation high </td><Td> 5uL </Td></tr><br />
<tr><td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><BR><br />
12. Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(+) plate and cultured at 37℃for 16 hours.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 11th</h2><br />
<br><br />
1. Single colony isolation <br />
<br><br />
Single colonies were isolated from the plate prepared on 9/11 and cultured in 2.5mL LB Chloramphenicol(+) medium at 37℃ for 16 hours.<br />
<br><br><br />
2. Isolation of the candidate pSB1C3-UAS DNA<br />
<br><br />
The candidate pSB1C3-UAS DNA was purified by QIA prep Spin Miniprep Kit.<br />
<br><Br><br />
3. The purified candudate pSB1C3-UAS was digested with BglII for 2 hours.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS-1, -2, -3, -4 </Td><Td> 5uL </Td></Tr><br />
<tr><td> 3buffer(NEB) </td><Td> 2uL </Td></tr><br />
<Tr><Td> BglⅡ </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><Td> 12.5uL </Td></tr><br />
<td> Total </td><Td> 20uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were applied to agarose gel electrophoresis as shown below. Left to right: pSB1C3-1 uncut, pSB1C3 cut, -2 uncut, -2 cut, -3 uncut, -3 cut, -4 uncut, -4 cut<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/0/0c/0911akit.png" width="500" height="300"><br />
<br><br><br />
Results: All DNAs examined had a single BglII site, indicating that the pSB1C3-UAS DNA was successfully constructed.<br />
<br><br><br />
The BglII-digested pSB1C3-UAS DNA was purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
4. Digestion of pSB1C3 (PCR) with XbaⅠ and SpeⅠ<br />
<br><br />
PCR-amplified pSB1C3 DNA was double digested with XbaⅠ and SpeⅠ at 37℃ for 2 hours. <br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> XbaⅠ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.5uL </Td></Tr><Tr><br />
<Td> CIP </Td><Td> 0.5uL </Td></Tr><Tr><br />
<Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><Td> 3uL </Td></tr><br />
<td> Total </td><Td> 40uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were purified by QIA quick Gel Extraction Kit<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/da/0911kit.png" width="500" height="300"><br />
<br><br><br />
5. Insertion of GAL4, HS promoter or Act5C promoter-enhancer into the pSB1C3DNA<br />
<br><br />
<br><br />
Ligation reactions were carried out under conditions described below at 16℃ for 1hour .<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (vector) (digested with XbaⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (digestion with BglⅡ and SpeⅠ) </td><Td> 2.5uL </Td></tr><br />
<Tr><Td> HS promoter or Act5C promoter-enhancer (digested with XbaⅠ and BamHⅠ) </Td><Td> 2.5uL </Td></Tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCR products) (digested with XbaⅠ and SpeⅠ) </Td><Td> 2uL </Td></Tr><br />
<tr><td> GAL4 (digested with BglⅡ and SpeⅠ) </td><Td> 2.5uL </Td></tr><br />
<Tr><Td> HS promoter or Act5C promoter-enhancer (XbaⅠ and BamHⅠ) </Td><Td> 1.5uL </Td></Tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
13. Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(+) plate and cultured at 37℃for 16 hours.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<h2>September 12th</h2><br />
<br><br />
<strong>1-2-7 Purification of the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs</strong><br />
<br><br />
We purified the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs by QIA prep Spin Miniprep Kit from E. coli cultured in LB medium for 16 hours (9/11). <br />
<br><br><br />
<strong>1-1-47 Digestion of pSB1C3-UAS DNA by Spe1</strong><br />
<br><br />
We incubated BglⅡ-digested pSB1C3-UAS (9/11) DNA with SpeⅠfor 2 hours. The reaction conditions are shown below.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS-1, -2 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 1uL </Td></Tr><br />
<td> 100×BSA </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 3.5uL </Td></tr><br />
<td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA were applied to agarose gel electrophoresis. We isolated DNA from the gel by using QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of Agarose gel.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/a/ab/0912kit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-1-48 and 2-2-6 Ligation</strong><br />
<br><br />
DNA fragment carrying EGFP or LacZ was ligated into the BglII and SpeI digested pSB1C3-UAS DNA .for 1hour at 16℃.<br />
<br><br><br />
Ligation reactions are listed below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS (double digested with BglII and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> EGFP fragments or LacZ fragments </td><Td> 2uL </Td></tr><br />
<Tr><Td> EGFP or LacZ </Td><Td> 2uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 2.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 2uL </Td></tr><tr><br />
<td> Total </td><Td> 7.5uL </Td></tr><br />
</Table><br />
<br><BR><br />
We ligated DNA fragment carrying GAL4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA for 1hour at 16℃.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCR) (double digested with XbaⅠand SpeI) </Td><Td> 2uL </Td></Tr><br />
<tr><td> GAL4(double digested with BglⅡ and SpeⅠ) </td><Td> 2uL </Td></tr><br />
<Tr><Td> HS fragments or Act5c fragments (double digested with XbaⅠand BamHⅠ) </Td><Td> 2uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-49 and 2-2-7 Transformation of E. coli by Ligation products</strong><br />
<br><br />
We did transformation of E. coli Xl-1 blue with each of ligation products. Finally, we spread them on the LB Chloramphenicol(+) plate and incubated for 16 hours at 37℃.<br />
<br />
<br><br><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week1p
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2012-09-26T06:14:27Z
<p>Kazuko: </p>
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<div id="MIGI"><br />
<h2>August 23rd</h2><br />
<br><br />
<strong>1-1-1 and 2-1-1 Transformation of E. coli with pEGFP-C2 DNA and pAct5C-GAL4 DNA</strong><br />
<br><br />
E. coli transformed with pEGFP-C2 was spread on the LB Kanamycin(+) plate and that transformed with pAct5C GAL4 was spread on the LB ampicillin(+) plate.<br />
<br />
<br><br><br />
<br />
<br />
<h2>August 24th</h2><br />
<br><br />
<strong>1-1-2 and 2-1-2</strong><br />
<br><br />
The appropriate colonies were picked up and cultured in the LB medium containing the appropriate antibiotics.<br />
<br><br><br />
<br />
<br />
<h2>August 25th</h2><br />
<br><br />
<strong>1-1-3 and 2-1-3 Purification of the pEGFP-C2 DNA and the pAct5C-GAL4 DNA</strong><br />
<br><br />
These plasmid DNAs were purified from E. coli by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
<strong>1-1-4 Digestion of pEGFP-C2 DNA with BamHⅠ and BglⅡt</strong><br />
<br><br />
Restriction enzyme digestion was carried out in the following reaction<br />
<br><br><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Total DNA amount </Td><Td> 500ng </Td><Td> 1ug </Td></Tr><br />
<tr><td> pEGFP-C </td><td> 3uL </td><Td> 6uL </Td></tr><br />
<Tr><Td> H buffer(TOYOBO) </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> BamHⅠ(TOYOBO) </td><td> 1uL </td><Td> 1uL </Td></tr><tr><br />
<td> BglⅡ(TOYOBO) </td><td> 1uL </td><Td> 1uL </Td></tr><tr><br />
<td> 100×BSA </td><td> 0.5uL </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 39.5uL </td><Td> 36.5uL </Td></tr><tr><br />
<td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><br><br />
<strong>1-1-5 Agarose gel electrophoresis of the digested DNA </strong><br />
<br><br />
The digested DNA was applied to the agarose gel electrophoresis and linearized DNA was purified by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photograph of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d0/0825kit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-1-6 Digestion of pSB1C3DNA with EcoRⅠ and SpeⅠ</strong><br />
<br><br />
pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ at 37℃ for 16 hours.<br />
<br><BR><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA sample </td><td> 23uL </td></tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<tr><td> EcoRⅠ-HF(NEB) </td><td> 1uL </td></tr><tr><br />
<td> SpeⅠ(NEB) </td><td> 1.5uL </td></tr><br />
<td> 100×BSA </td><td> 0.5uL </td></tr><br />
<td> dH<sub>2</sub>O </td><td> 19uL </td></tr><br />
<td> Total <td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
<br />
<strong>1-1-7 PCR amplification of the UAS region and pSB1C3 DNA</strong><br />
<br><br />
DNA fragments containing UAS was amplified from the pUAST DNA and the BglⅡ site-containing pSB1C3 was also amplified by PCR.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA template </td><td> 0.5uL </td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 10uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 10uL </td></tr><tr><br />
<td> 25mM MgSO4 </td><td> 3.2uL </td></tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<td> KOD plus </td><td> 2uL </td></tr><br />
<td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<td> Total <td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Circle </Td></Tr><br />
<tr><td> 95℃</td><td> 2min</td><Td></Td></tr><br />
<Tr><Td> 95℃</Td><Td> 15sec</Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃</td><td> 30min(UAS) or 2min10sec(pSB1C3)</td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃</td><td> 2min30sec</td><Td> 25 cycle </Td></tr><br />
<td> 68℃</td><td> 2min30sec</td><Td></Td></tr><br />
<td> 14℃</td><td> ∞</td><Td></Td></tr><br />
</Table><br />
<br><br><br />
<br />
<br />
<br />
<br />
<br />
<h2>August 26th</h2><br />
<br><br />
<strong>1-1-8 Purification of the digested pSB1C3</strong><br />
<br><br />
pSB1C3 DNA double-digested with EcoRⅠ and SpeⅠ(prepared on 8/25) was applied to the agarose gel electrophoresis. The DNA fragment of interest was purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/0826.png" width="500" height="300"><br />
<br><br />
<br><br />
<h2>August 27th</h2><br />
<br><br />
<strong>1-1-9 Removing the multi-cloning site of the pEGFP-C2</strong><br />
<br><br />
pEGFP-C2 DNA digested with BglⅡ and BamHⅠwas self ligated in the following reaction.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pEGFP-C2(cut) </Td><Td> 1uL </Td></Tr><br />
<tr><td> Ligation high </td><td> 2.5uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 5uL </td></tr><tr><br />
<td> Total <td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-10</strong><br />
<br><br />
The ligated products were transformed into the E. coli XL-1 blue and the E. coli was apreaded on the LB Kanamycin(+) plate and cultured at 37℃ for 16 hours.<br />
<br><br><br />
<strong>1-1-11 Agarose gel electrophoresis of the DNA fragments containing UAS and the PSR products from the pSB1C3 </strong><br />
<br><br />
PCRproducts prepared on August 25th were applied to the agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/e6/0826bkit.png" width="500" height="300"><br />
<br><br><br />
Since the expected DNA fragments were detected, DNA was purified from the agarose gel by QIA quick Gel Extraction Kit. The purified DNAs were applied to the agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/99/0826ckit.png" width="500" height="300"><br />
<br><br><br />
<br />
<br />
<br />
<br />
<h2>August 28th</h2><br />
<br><br />
<strong>1-1-12 Single colony isolation from the E. coli transformed with pEGFP-C2 DNA</strong><br />
<br><br />
Single colony isolationwas carried out from the plate prepared on 8/27and cultured in 2.5mL LB Kanamycin(+) medium at 37℃ for 16 hours.<br />
<br><br><br />
<strong>2-1-4 Transformation of E. coli with pGaTB DNA or pAct5C-GAL4 DNA</strong><br />
<br><br />
E. coli Xl-1 blue was transformed with pGaTBDNA or pAct5C-GAL4 DNA and cultured on LB ampicillin(+) plate for 16 hours at 37℃.<br />
<br><br><br />
<br />
<br />
<br />
<h2>August 29th</h2><br />
<br><br />
<strong>2-1-5 Single colony isolation from the E. coli transformed with pGaTB DNA or pAct5C-GAL4 DNA</strong><br />
<br><br />
From the plate prepared on 8/28 single colonies were isolated and cultured in 2.5mL LB ampicillin(+) medium for 16 hours at 37℃.<br />
<br><br><br />
<strong>1-1-13 Purification of pEGFP-C2 DNA</strong><br />
<br><br />
pEGFP-C2 DNA was purified from E. coli prepared on 8/28 by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
<strong>1-1-14 Cut check of the pEGFP-C2 DNA</strong><br />
<br><br />
The purified pEGFP-C2(1-1-13) was digested with XhoⅠ and PstⅠ in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pEGFP-C2-1, -2, -3, -4 </Td><Td> 1uL </Td></Tr><br />
<tr><td> 10×H buffer(TOYOBO) </td><td> 0.5uL </td></tr><tr><br />
<tr><td> XhoⅠ </td><td> 0.1uL </td></tr><tr><br />
<tr><td> PstⅠ </td><td> 0.1uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 3.3uL </td></tr><tr><br />
<td> Total <td> 5uL </td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were applied to the agarose gel electrophoresis.as shown below. <br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/97/0828kit.png" width="500" height="300"><br />
<br><br><br />
Results: The DNAs were undigested by these enzymes, confirming that the multi-cloning site of the pEGFP-C2 was successfully removed.<br />
<br><br><br />
<strong>1-1-15 PCR amplification of the DNA fragments containing UAS and EGFP</strong><br />
<br><br />
Since we found that the previously used primers for PCR were wrong, PCR amplification of the DNA fragments containing UAS and EGFP region were carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 10uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 10uL </Td></Tr><br />
<Tr><Td> 25mM MgSO4 </Td><Td> 3.2uL </Td></Tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<Tr><td> KOD plus </td><td> 2uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<Tr><td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 1min(EGFP) </td><Td> 25 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 1min(EGFP) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
Agarose gel electrophoresis of the PCR products<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d2/0829b.png" width="500" height="300"><br />
<br><br><br />
Results: DNA fragments containing EGFP were successfully amplified, but not for the UAS.<br />
<br><br><br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week2p">>>>>>>>>>WEEK2</a></div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week1p
Team:KIT-Kyoto/Notebook-week1p
2012-09-26T06:10:18Z
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<div id="MIGI"><br />
<h2>August 23rd</h2><br />
<br><br />
<strong>1-1-1 and 2-1-1 Transformation of E. coli with pEGFP-C2 DNA and pAct5C-GAL4 DNA</strong><br />
<br><br />
E. coli transformed with pEGFP-C2 was spread on the LB Kanamycin(+) plate and that transformed with pAct5C GAL4 was spread on the LB ampicillin(+) plate.<br />
<br />
<br><br><br />
<br />
<br />
<h2>August 24th</h2><br />
<br><br />
<strong>1-1-2 and 2-1-2</strong><br />
<br><br />
The appropriate colonies were picked up and cultured in the LB medium containing the appropriate antibiotics.<br />
<br><br><br />
<br />
<br />
<h2>August 25th</h2><br />
<br><br />
</strong>1-1-3 and 2-1-3 Purification of the pEGFP-C2 DNA and the pAct5C-GAL4 DNA</strong><br />
<br><br />
These plasmid DNAs were purified from E. coli by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
<strong>1-1-4 Digestion of pEGFP-C2 DNA with BamHⅠ and BglⅡt</strong><br />
<br><br />
Restriction enzyme digestion was carried out in the following reaction<br />
<br><br><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Total DNA amount </Td><Td> 500ng </Td><Td> 1ug </Td></Tr><br />
<tr><td> pEGFP-C </td><td> 3uL </td><Td> 6uL </Td></tr><br />
<Tr><Td> H buffer(TOYOBO) </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> BamHⅠ(TOYOBO) </td><td> 1uL </td><Td> 1uL </Td></tr><tr><br />
<td> BglⅡ(TOYOBO) </td><td> 1uL </td><Td> 1uL </Td></tr><tr><br />
<td> 100×BSA </td><td> 0.5uL </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 39.5uL </td><Td> 36.5uL </Td></tr><tr><br />
<td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><br><br />
<strong>1-1-5 Agarose gel electrophoresis of the digested DNA </strong><br />
<br><br />
The digested DNA was applied to the agarose gel electrophoresis and linearized DNA was purified by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photograph of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d0/0825kit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-1-6 Digestion of pSB1C3DNA with EcoRⅠ and SpeⅠ</strong><br />
<br><br />
pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ at 37℃ for 16 hours.<br />
<br><BR><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA sample </td><td> 23uL </td></tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<tr><td> EcoRⅠ-HF(NEB) </td><td> 1uL </td></tr><tr><br />
<td> SpeⅠ(NEB) </td><td> 1.5uL </td></tr><br />
<td> 100×BSA </td><td> 0.5uL </td></tr><br />
<td> dH<sub>2</sub>O </td><td> 19uL </td></tr><br />
<td> Total <td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
<br />
<strong>1-1-7 PCR amplification of the UAS region and pSB1C3 DNA</strong><br />
<br><br />
DNA fragments containing UAS was amplified from the pUAST DNA and the BglⅡ site-containing pSB1C3 was also amplified by PCR.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA template </td><td> 0.5uL </td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 10uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 10uL </td></tr><tr><br />
<td> 25mM MgSO4 </td><td> 3.2uL </td></tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<td> KOD plus </td><td> 2uL </td></tr><br />
<td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<td> Total <td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Circle </Td></Tr><br />
<tr><td> 95℃</td><td> 2min</td><Td></Td></tr><br />
<Tr><Td> 95℃</Td><Td> 15sec</Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃</td><td> 30min(UAS) or 2min10sec(pSB1C3)</td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃</td><td> 2min30sec</td><Td> 25 cycle </Td></tr><br />
<td> 68℃</td><td> 2min30sec</td><Td></Td></tr><br />
<td> 14℃</td><td> ∞</td><Td></Td></tr><br />
</Table><br />
<br><br><br />
<br />
<br />
<br />
<br />
<br />
<h2>August 26th</h2><br />
<br><br />
<strong>1-1-8 Purification of the digested pSB1C3</strong><br />
<br><br />
pSB1C3 DNA double-digested with EcoRⅠ and SpeⅠ(prepared on 8/25) was applied to the agarose gel electrophoresis. The DNA fragment of interest was purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/0826.png" width="500" height="300"><br />
<br><br />
<br><br />
<h2>August 27th</h2><br />
<br><br />
<strong>1-1-9 Removing the multi-cloning site of the pEGFP-C2</strong><br />
<br><br />
pEGFP-C2 DNA digested with BglⅡ and BamHⅠwas self ligated in the following reaction.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pEGFP-C2(cut) </Td><Td> 1uL </Td></Tr><br />
<tr><td> Ligation high </td><td> 2.5uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 5uL </td></tr><tr><br />
<td> Total <td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-10</strong><br />
<br><br />
The ligated products were transformed into the E. coli XL-1 blue and the E. coli was apreaded on the LB Kanamycin(+) plate and cultured at 37℃ for 16 hours.<br />
<br><br><br />
<strong>1-1-11 Agarose gel electrophoresis of the DNA fragments containing UAS and the PSR products from the pSB1C3 </strong><br />
<br><br />
PCRproducts prepared on August 25th were applied to the agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/e6/0826bkit.png" width="500" height="300"><br />
<br><br><br />
Since the expected DNA fragments were detected, DNA was purified from the agarose gel by QIA quick Gel Extraction Kit. The purified DNAs were applied to the agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/99/0826ckit.png" width="500" height="300"><br />
<br><br><br />
<br />
<br />
<br />
<br />
<h2>August 28th</h2><br />
<br><br />
<strong>1-1-12 Single colony isolation from the E. coli transformed with pEGFP-C2 DNA</strong><br />
<br><br />
Single colony isolationwas carried out from the plate prepared on 8/27and cultured in 2.5mL LB Kanamycin(+) medium at 37℃ for 16 hours.<br />
<br><br><br />
<strong>2-1-4 Transformation of E. coli with pGaTB DNA or pAct5C-GAL4 DNA</strong><br />
<br><br />
E. coli Xl-1 blue was transformed with pGaTBDNA or pAct5C-GAL4 DNA and cultured on LB ampicillin(+) plate for 16 hours at 37℃.<br />
<br><br><br />
<br />
<br />
<br />
<h2>August 29th</h2><br />
<br><br />
<strong>2-1-5 Single colony isolation from the E. coli transformed with pGaTB DNA or pAct5C-GAL4 DNA</strong><br />
<br><br />
From the plate prepared on 8/28 single colonies were isolated and cultured in 2.5mL LB ampicillin(+) medium for 16 hours at 37℃.<br />
<br><br><br />
<strong>1-1-13 Purification of pEGFP-C2 DNA</strong><br />
<br><br />
pEGFP-C2 DNA was purified from E. coli prepared on 8/28 by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
<strong>1-1-14 Cut check of the pEGFP-C2 DNA</strong><br />
<br><br />
The purified pEGFP-C2(1-1-13) was digested with XhoⅠ and PstⅠ in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pEGFP-C2-1, -2, -3, -4 </Td><Td> 1uL </Td></Tr><br />
<tr><td> 10×H buffer(TOYOBO) </td><td> 0.5uL </td></tr><tr><br />
<tr><td> XhoⅠ </td><td> 0.1uL </td></tr><tr><br />
<tr><td> PstⅠ </td><td> 0.1uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 3.3uL </td></tr><tr><br />
<td> Total <td> 5uL </td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were applied to the agarose gel electrophoresis.as shown below. <br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/97/0828kit.png" width="500" height="300"><br />
<br><br><br />
Results: The DNAs were undigested by these enzymes, confirming that the multi-cloning site of the pEGFP-C2 was successfully removed.<br />
<br><br><br />
<strong>1-1-15 PCR amplification of the DNA fragments containing UAS and EGFP</strong><br />
<br><br />
Since we found that the previously used primers for PCR were wrong, PCR amplification of the DNA fragments containing UAS and EGFP region were carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 10uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 10uL </Td></Tr><br />
<Tr><Td> 25mM MgSO4 </Td><Td> 3.2uL </Td></Tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<Tr><td> KOD plus </td><td> 2uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<Tr><td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 1min(EGFP) </td><Td> 25 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 1min(EGFP) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
Agarose gel electrophoresis of the PCR products<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d2/0829b.png" width="500" height="300"><br />
<br><br><br />
Results: DNA fragments containing EGFP were successfully amplified, but not for the UAS.<br />
<br><br><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week3p
Team:KIT-Kyoto/Notebook-week3p
2012-09-26T05:33:10Z
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<td width="800px" height="510px" valign="top"><br />
<div id="MIGI"><br />
<h2>September 7th</h2><br />
<br><br />
1. PCR amplification of UAS, UAS-TNFAIP3, pSB1C3, GAL4 DNA<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.25uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 34.15uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
2. PCR products were applied to the agarose gel electrophoresis<br />
<br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 as shown below<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/0907akit.png" width="500" height="300"><br />
<br><br><br />
Results: no clearly amplified bands were detected.<br />
<br><br><br />
3. Re-estimation of the concentration of pSB1C3 and GAL4 DNA used for ligation on 9/5, 6<br />
<br><br><br />
4. SB1C3 and GAL4 DNA were applied to the agarose gel electrophoresis<br />
<br><br />
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker - 2uL, DNA 0.1uL, 1kbmarker 1uL<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/68/0907bkit.png" width="500" height="300"><br />
<br><br><br />
From these results, concentration of the pSB1C3 DNA was estimated to be 10ng/uL.<br />
<br><br />
<br><br />
GAL4<br />
<br><br />
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker 2uL, DNA 0.1uL, 1kbmarker 1uL<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/0907ckit.png" width="500" height="300"><br />
<br><br><br />
However no GAL DNA was detected. We lost the sample somewhere by some mistakes.<br />
<br><br><br />
<br />
<br />
<h2>September 8th</h2><br />
<br><br />
1. PCR amplification of UAS, UAS-TNFAIP3, pSB1C3 and GAL4 DNA<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td></td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
2. PCRproducts applied to the agarose gel electrophoresis<br />
<br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 10 uL each<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/be/0908kit.png" width="500" height="300"><br />
<br><br><br />
Only the PCR products for pSB1C3 was detectable.<br />
<br><br />
<br><br />
3. We repeated PCR for UAS and GAL4DNA by changing the annealing temperature<br />
<br><br />
(-1 indicates 55℃、-2 indicates 58℃)<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 55℃(-1) or 58℃(-2) </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<h2>September 9th</h2><br />
<BR><br />
1. PCRproducts were electrophoreses.<br />
<br><br />
Left to right: UAS-1, UAS-2, GAL4-1, GAL4-2 10 uL each<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/1/10/0909akit.png" width="500" height="300"><br />
<br><br><br />
We finally succeeded the amplification of the UAS fragments.<br />
<br><br><br />
2. Purification of pSB1C3 and UAS-1-PCR products<br />
<br><br />
PCR products were purified by High Pure PCR Product Kit<br />
<br><br><br />
3. The purified UAS fragments were digested with EcoRⅠ and SpeⅠ in the following reaction<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> UAS </Td><Td> 40uL </Td></Tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<Tr><td> SpeⅠ(NEB </td><td> 0.5uL </td></tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 3.5uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
4. The digested UAS DNA was applied to the agarose gel electrophoresis and purified from the gel by QIA quick Gel Extraction Kit<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/91/0909bkit.png" width="500" height="300"><br />
<br><br><br />
5. PCR amplification of GAL4<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs <td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min10sec </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 2min10sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
6. PCR products were applied to the agarose gel electrophoresis as shown below.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/bb/0909ckit.png" width="500" height="300"><br />
<br><br><br />
7. pSB1C3 and UAS fragments were ligated in the following reactions.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (EcoRⅠ and SpeⅠ digested) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> UAS (EcoRⅠ and SpeⅠ digested) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3uL </Td></Tr><br />
<Tr><td> Ligation high </td><td> 2.5uL </td></tr><br />
<Tr><Td> Total </Td><Td> 7.5uL </Td></Tr><br />
</Table><br />
<br><BR><br />
The following reaction were carried out as a negative control. <br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (EcoRⅠ and SpeⅠ digested)</Td><Td> 1uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 4uL </Td></Tr><br />
<Tr><td> Ligation high </td><td> 2.5uL </td></tr><br />
<Tr><Td> Total </Td><Td> 7.5uL </Td></Tr><br />
</Table><br />
<br><BR><br />
8. 7 ligation products were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(+) plate and incubated at 37℃ for 16 hours.<br />
<br><br><br />
<br />
<h2>September 10th</h2><br />
<br><br />
At 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP-TNFAIP3 DNA was added to the well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 hours at 25 ℃.<br />
<br><br />
<br><br />
1. Single colony isolation of ligationproducts<br />
<br><br />
Ligation product prepared on 9/9 (UAS and pSB1C3) gave us some colonies and negative control sample gave us no colony. We therefore picked up four independent colonies and cultured them in 2.5mL LB Chloramphenicol (+) medium at 37℃ for 16 hours.<br />
<br><br><br />
2. Digestion of HS promoter fragment and Act5C promoter with XbaⅠ and BamHⅠ<br />
<br><br />
DNA fragments carrying HS promoter (prepared on 9/5) and those carrying Act5C promoter (prepared on 9/3) were digested with XbaⅠ and BamHⅠ.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> HS or Act5c </Td><Td> 40uL </Td></Tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<Tr><td> XbaⅠ-HF(NEB) <td> 0.5uL </td></tr><br />
<Tr><Td> BamHⅠ(NEB )</Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3.5uL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 50uL </Td></Tr><br />
</Table><br />
<br><BR><br />
Digested fragments were purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/27/0910akit.png" width="500" height="300"><br />
<br><br><br />
3. PCR amplification of GAL4 fragments<br />
<br><br />
We tried PCR under four different conditions described below. <br />
<Br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><Td> G-1 and G-2 </Td><Td> G-3 and G-4 </Td></Tr><br />
<tr><td> DNA template </td><td> 1uL </td><Td> 4uL </Td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 5uL </td><Td> 5uL </Td></tr><tr><br />
<td> 25mM MgSO<sub>4</sub> </td><td> 1.6uL </td><Td> 1.6uL </Td></tr><br />
<tr><td> 10P 5’ primer </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> 10P 3’ primer </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> KOD plus </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td><Td> 31.4uL </Td></tr><br />
<tr><td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 57℃(G-1 and G-3) or 59℃(G-2 and G-4) </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min10sec </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 2min10sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
4. PCR products were applied to agarose gel electrophoresis <br />
<br><br />
From left to right: 10uL each G-1, G-2, G-3, G-4<br />
<br><br />
<br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/eb/0910bkit.png" width="500" height="300"><br />
<br><br><br />
We found that GAL4 sequence was properly amplified under G-1 and G-2conditions.<br />
<brr><br />
5. PCR products were purified by High Pure PCR Product Kit.<br />
<br><br />
6. Digestion of pSB1C3 DNA with EcoRⅠ and SpeⅠ<br />
<br><br />
PCR-amplified pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ for 2 hours at 37℃.<br />
<br><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3(PCR) </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> SpeⅠ(NEB) </td><Td> 0.5uL </Td></tr><tr><br />
<td> 2100×BSA </td><Td> 0.5uL </Td></tr><br />
<tr><td> dH<sub>2</sub>O <Td> 3.5uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
7. Digestion of EcoRI-digested UAS fragment by BglⅡ<br />
<br><br />
BglⅡ-digestion was carried out for 2 hours at 37℃ under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> UAS(EcoRⅠ-digested) </Td><Td> 13uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><Td> 2uL </Td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O <Td> 2.5uL </Td></tr><br />
<tr><td> Total </td><Td> 20uL </Td></tr><br />
</Table><br />
<br><BR><br />
8. Purification of restriction enzyme-digested pSB1C3 DNA and UAS fragment<br />
<br><br />
restriction enzyme-digested pSB1C3 DNA and UAS fragment (prepared on 9/10) were purified by QIA quick Gel Extraction Kit. <br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/86/0910ckit.png" width="500" height="300"><br />
<br><br><br />
9. PCR-amplified GAL4 fragments were digested with XbaⅠ and SpeⅠ under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 20uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 4uL </Td></tr><br />
<Tr><Td> XbaⅠ(NEB) </Td><Td> 0.7uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.7uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.4uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 14.6uL </Td></tr><br />
<tr><td> Total </td><Td> 40uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA was applied to agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/3/39/0910dkit.png" width="500" height="300"><br />
<br><br><br />
Results: XbaⅠ-digestion of GAL4 fragment gave two DNA fragments, indicating that GAL4 sequence contains XbaⅠ site.<br />
<br><br><br />
10. Digestion of GAL4 sequence with BglⅡ and SpeⅠ<br />
<br><br />
Digestion was carried out st 37℃ for 1hour under the conditions described below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 4.5uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA was purified by High Pure PCR Product Kit was further digested with Spe I at 37℃ for 1hour.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4(Bgl Ⅱ cut) </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 4uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested fragments were purified by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/0910ekit.png" width="500" height="300"><br />
<br><br><br />
11. Insertion of GAL4 fragments and HS promoter or Act5C promoter-enhancer and UASfragment and EGFP sequence or LacZ sequence into the pSB1C3DNA<br />
<br><br><br />
Ligation reactions were carried out at 16℃ for 1hour under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCRproducts)(double digested with EcoRⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> UAS (double digested with EcoRⅠ and BglⅡ) </td><Td> 2uL </Td></tr><br />
<Tr><Td> EGFP or LacZ </Td><Td> 2uL </Td></Tr><br />
<tr><td> Ligation high </td><Td> 5uL </Td></tr><br />
<tr><td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (vector DNA) (double digested with XbaⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (double digested with BglⅡ and SpeⅠ) </td><Td> 2uL </Td></tr><br />
<Tr><Td>HS or Act5c (double digested with XbaⅠ and BamHⅠ)</Td><Td> 2uL </Td></Tr><br />
<tr><td> Ligation high </td><Td> 5uL </Td></tr><br />
<tr><td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><BR><br />
12. Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(+) plate and cultured at 37℃for 16 hours.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 11th</h2><br />
<br><br />
1. Single colony isolation <br />
<br><br />
Single colonies were isolated from the plate prepared on 9/11 and cultured in 2.5mL LB Chloramphenicol(+) medium at 37℃ for 16 hours.<br />
<br><br><br />
2. Isolation of the candidate pSB1C3-UAS DNA<br />
<br><br />
The candidate pSB1C3-UAS DNA was purified by QIA prep Spin Miniprep Kit.<br />
<br><Br><br />
3. The purified candudate pSB1C3-UAS was digested with BglII for 2 hours.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS-1, -2, -3, -4 </Td><Td> 5uL </Td></Tr><br />
<tr><td> 3buffer(NEB) </td><Td> 2uL </Td></tr><br />
<Tr><Td> BglⅡ </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><Td> 12.5uL </Td></tr><br />
<td> Total </td><Td> 20uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were applied to agarose gel electrophoresis as shown below. Left to right: pSB1C3-1 uncut, pSB1C3 cut, -2 uncut, -2 cut, -3 uncut, -3 cut, -4 uncut, -4 cut<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/0/0c/0911akit.png" width="500" height="300"><br />
<br><br><br />
Results: All DNAs examined had a single BglII site, indicating that the pSB1C3-UAS DNA was successfully constructed.<br />
<br><br><br />
The BglII-digested pSB1C3-UAS DNA was purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
4. Digestion of pSB1C3 (PCR) with XbaⅠ and SpeⅠ<br />
<br><br />
PCR-amplified pSB1C3 DNA was double digested with XbaⅠ and SpeⅠ at 37℃ for 2 hours. <br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> XbaⅠ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.5uL </Td></Tr><Tr><br />
<Td> CIP </Td><Td> 0.5uL </Td></Tr><Tr><br />
<Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><Td> 3uL </Td></tr><br />
<td> Total </td><Td> 40uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were purified by QIA quick Gel Extraction Kit<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/da/0911kit.png" width="500" height="300"><br />
<br><br><br />
5. Insertion of GAL4, HS promoter or Act5C promoter-enhancer into the pSB1C3DNA<br />
<br><br />
<br><br />
Ligation reactions were carried out under conditions described below at 16℃ for 1hour .<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (vector) (digested with XbaⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (digestion with BglⅡ and SpeⅠ) </td><Td> 2.5uL </Td></tr><br />
<Tr><Td> HS promoter or Act5C promoter-enhancer (digested with XbaⅠ and BamHⅠ) </Td><Td> 2.5uL </Td></Tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCR products) (digested with XbaⅠ and SpeⅠ) </Td><Td> 2uL </Td></Tr><br />
<tr><td> GAL4 (digested with BglⅡ and SpeⅠ) </td><Td> 2.5uL </Td></tr><br />
<Tr><Td> HS promoter or Act5C promoter-enhancer (XbaⅠ and BamHⅠ) </Td><Td> 1.5uL </Td></Tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
13. Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(+) plate and cultured at 37℃for 16 hours.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<h2>September 12th</h2><br />
<br><br />
<strong>1-2-7 Purification of the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs</strong><br />
<br><br />
We purified the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs by QIA prep Spin Miniprep Kit from E. coli cultured in LB medium for 16 hours (9/11). <br />
<br><br><br />
<strong>1-1-47 Digestion of pSB1C3-UAS DNA by Spe1</strong><br />
<br><br />
We incubated BglⅡ-digested pSB1C3-UAS (9/11) DNA with SpeⅠfor 2 hours. The reaction conditions are shown below.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS-1, -2 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 1uL </Td></Tr><br />
<td> 100×BSA </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 3.5uL </Td></tr><br />
<td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA were applied to agarose gel electrophoresis. We isolated DNA from the gel by using QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of Agarose gel.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/a/ab/0912kit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-1-48 and 2-2-6 Ligation</strong><br />
<br><br />
DNA fragment carrying EGFP or LacZ was ligated into the BglII and SpeI digested pSB1C3-UAS DNA .for 1hour at 16℃.<br />
<br><br><br />
Ligation reactions are listed below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS (double digested with BglII and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> EGFP fragments or LacZ fragments </td><Td> 2uL </Td></tr><br />
<Tr><Td> EGFP or LacZ </Td><Td> 2uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 2.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 2uL </Td></tr><tr><br />
<td> Total </td><Td> 7.5uL </Td></tr><br />
</Table><br />
<br><BR><br />
We ligated DNA fragment carrying GAL4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA for 1hour at 16℃.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCR) (double digested with XbaⅠand SpeI) </Td><Td> 2uL </Td></Tr><br />
<tr><td> GAL4(double digested with BglⅡ and SpeⅠ) </td><Td> 2uL </Td></tr><br />
<Tr><Td> HS fragments or Act5c fragments (double digested with XbaⅠand BamHⅠ) </Td><Td> 2uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-49 and 2-2-7 Transformation of E. coli by Ligation products</strong><br />
<br><br />
We did transformation of E. coli Xl-1 blue with each of ligation products. Finally, we spread them on the LB Chloramphenicol(+) plate and incubated for 16 hours at 37℃.<br />
<br />
<br><br><br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week4p">>>>>>>>>>WEEK4</a></div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week2p
Team:KIT-Kyoto/Notebook-week2p
2012-09-26T05:24:32Z
<p>Kazuko: </p>
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<h2>August 31st</h2><br />
<br><br />
1. PCR amplification of DNA fragments containing UAS and Heat Shock promoter<br />
<br><br />
We have carried out the PCR reaction for UAS again. PCR amplification of Heat Shock promoter from pCasPer DNA was also carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 10uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 10uL </Td></Tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 3.2uL </Td></Tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<Tr><td> KOD plus </td><td> 2uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<Tr><td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature</Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
2. Purification of pAct5C DNA and pGaTB DNA<br />
<br><br />
pAct5C DNA and pGaTB DNA was purified from E. coli prepared on 8/29by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
<br />
<h2>September 1st</h2><br />
<br><br />
1. Agarose gel electrophoresis of PCR produsts and the purified pAct5C DNA and pGaTB DNA<br />
<br><br />
Agarose gel image is shown below. Left to right: HS promoter 10uL, UAS fragment 10uL, pAct5C DNA-1, pGaTB DNA-1, pGaTB DNA-2, pAct5C DNA<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/b/b0/0901akit.png" width="500" height="300"><br />
<br><br><br />
2. Expected bands for pAct5C DNA and pGaTB DNA were detected on the gel, but band for UAS fragment was not.<br />
<br><br><br />
<br />
<h2>September 3rd</h2><br />
<BR><br />
1. PCR amplification of DNA fragments containing GAL4, Act5C promoter and LacZ sequence<br />
<br><br />
pGaTBDNA was used as a template to amplify GAL4 sequence. pAct5C-LacZ DNA was used to amplify Act5C promoter-enhancer region and LacZ. <br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA template </td><td> 0.2uL </td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 5uL </td></tr><tr><br />
<td> 25mM MgSO<sub>4</sub> </td><td> 1.6uL </td></tr><tr><br />
<td> 10P 5’ primer </td><td> 1.5uL </td></tr><tr><br />
<td> 10P 3’ primer </td><td> 1.5uL </td></tr><tr><br />
<td> KOD plus </td><td> 1u L</td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 34.2uL </td></tr><tr><br />
<td> Total <td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Circle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 55℃(GAL4) and 58℃(Except for GAL4) </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min30sec </td><Td> 25 cycle </Td></tr><br />
<td> 68℃ </td><td> 2min30sec </td><Td></Td></tr><br />
<td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><br><br />
2. 1 Agarose gel electrophoresis of the PCRproducts<br />
<br><br />
Left to right: 1kb ladder marker 5uL GAL4 fragments 10uL Act5C promoter-enhancer 10uL LacZ sequence <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/c/ca/0903kit.png" width="500" height="300"><br />
<br><br><br />
Results: Expected PCR products were all detected on the gel.<br />
<br><br />
These PCR products were purified by High Pure PCR Product Kit (Roche).<br />
<br><br><br />
<br />
<h2>September 4th</h2><br />
<br><br />
1. Digestion of GAL4 fragments by XbaⅠ and SpeⅠ<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 fragments prepared on 9/3 </Td><Td> 40uL </Td></Tr><br />
<tr><td> M buffer(TOYOBO) </td><td> 5uL </td></tr><br />
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> SpeⅠ(TOYOBO) </td><td> 0.5uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 4uL </td></tr><tr><br />
<td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
2. Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ<br />
<br><br />
pSB1C3 DNA prepared on 8/27, LacZ fragment prepared on 9/3 and EGFP fragments were digested with BglⅡ in the following conditions.<br />
<br><br />
<br><br />
3. Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ<br />
<br><br />
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> each DNA </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><td> 5uL </td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 4.5uL </td></tr><tr><br />
<td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ<br />
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.<br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 DNA </Td><Td> 23uL </Td></Tr><br />
<tr><td> M buffer(TOYOBO) </td><td> 10uL </td></tr><br />
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> SpeⅠ(TOYOBO) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> CIP </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><td> 64.5uL </td></tr><br />
<td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
4. Purification of GAL4 fragments<br />
<br><br />
After agarose gel electrophoresis, the digested GAL4 fragments were purified by QIA quick Gel Extraction Kit<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/b0/0904kit.png" width="500" height="300"><br />
<br><br><br />
5. Purification of BglII-digested LacZ fragments<br />
<br><br />
BglII-digested LacZ fragments were purified by High Pure PCR Product Kit.<br />
<br><br><br />
<br />
<h2>September 5th</h2><br />
<br><br />
1. Purification of EGFP fragments and HS promoter fragments<br />
<br><br />
EGFP fragments prepared on 8/29 and HS promoter fragments prepared on 8/31were purified by High Pure PCR Product Kit<br />
<br><br><br />
2. EGFP fragments and pSB1C3 DNA prepared on 8/27were digested with BglⅡ in the following reaction.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><td> EGFP </td><Td> pSB1C3 </Td></Tr><br />
<tr><td> DNA template </td><td> 40uL </td><Td> 23uL </Td></tr><br />
<Tr><Td> 3 buffer </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> BglⅡ </td><td> 0.5uL </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 4.5uL </td><Td> 21.5uL </Td></tr><br />
<td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
3. Purification of pSB1C3DNA digested with XbaⅠ and SpeⅠ<br />
<br><br />
Agarose gel electrophoresis image of the purified sample are shown below.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/3/3a/0905akit.png" width="500" height="300"><br />
<br><br><br />
4. SpeI digestion of the BglⅡ-digested LacZ, EGFP, pSB1C3 DNA<br />
<br><br />
SpeⅠ digestion was carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><td> 5uL </td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> CIP </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><td> 4uL </td></tr><br />
<td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
5. Ligation of pSB1C3 DNA and GAL4 fragment<br />
<br><br />
Ligation was carried out in the following reactions.<br />
<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (XbaⅠand SpeⅠdigested) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (XbaⅠ and SpeⅠ digested) </td><td> 1uL </td></tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3uL </Td></Tr><br />
<Tr><Td> Ligation high </Td><Td> 2.5uL </Td></Tr><br />
<td> Total </td><td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
6. The ligated products were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(+) plate<br />
<br><br><br />
7. Purification of pSB1C3, EGFP, LacZfragments double digested with BglⅡ and SpeⅠ<br />
<br><br />
The double digested samples were applied to the agarose gel electrophoresis and the DNA fragments were purified by QIA quick Gel Extraction Kit<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/63/0905bkit.png" width="500" height="300"><br />
<br><br><br />
<br />
<br />
<h2>September 6th</h2><br />
<br><br />
Transformation performed on September 5th was not successful, since we had no colony on the plate. <br />
<br><br><br />
1. PCR amplification of UAS , UAS-TNFAIP3, pSB1C3DNA<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.25uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 5uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 5uL </Td></Tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><tr><br />
<td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 34.15uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec</Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec</td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
2. PCR products were applied to the agarose gel electrophoresis<br />
<br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3<br />
<br><br><br />
<br />
3. Purifucation of pSB1C3 PCR products<br />
<br><br />
pSB1C3 PCR products were purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/5/53/0906kit.png" width="500" height="300"><br />
<br><br><br />
4. Ligation of pSB1C3 DNA and GAL4 fragment<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3(XbaⅠ and SpeⅠ cut) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> GAL4(XbaⅠ and SpeⅠ cut) </Td><Td> 1uL </Td></Tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 3uL </td></tr><br />
<Tr><Td> Ligation high </Td><Td> 2.5uL </Td></Tr><br />
<Tr><td> Total </td><td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
5. The ligation products were transformed into E. coli XL-1and spread on the LB Chloramphenicol(+) plate<br />
<br><br><br />
<br />
<br />
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<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week3p">>>>>>>>>>WEEK3</a></div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week1p
Team:KIT-Kyoto/Notebook-week1p
2012-09-26T05:19:02Z
<p>Kazuko: </p>
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<h2>August 23rd</h2><br />
<br><br />
Transformation of E. coli with pEGFP-C2 DNA and pAct5C-GAL4 DNA<br />
<br><br />
E. coli transformed with pEGFP-C2 was spread on the LB Kanamycin(+) plate and that transformed with pAct5C GAL4 was spread on the LB ampicillin(+) plate.<br />
<br />
<br><br><br />
<br />
<br />
<h2>August 24th</h2><br />
<br><br />
The appropriate colonies were picked up and cultured in the LB medium containing the appropriate antibiotics.<br />
<br><br><br />
<br />
<br />
<h2>August 25th</h2><br />
<br><br />
1. Purification of the pEGFP-C2 DNA and the pAct5C-GAL4 DNA<br />
<br><br />
These plasmid DNAs were purified from E. coli by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
2. Digestion of pEGFP-C2 DNA with BamHⅠ and BglⅡt<br />
<br><br />
Restriction enzyme digestion was carried out in the following reaction<br />
<br><br><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Total DNA amount </Td><Td> 500ng </Td><Td> 1ug </Td></Tr><br />
<tr><td> pEGFP-C </td><td> 3uL </td><Td> 6uL </Td></tr><br />
<Tr><Td> H buffer(TOYOBO) </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> BamHⅠ(TOYOBO) </td><td> 1uL </td><Td> 1uL </Td></tr><tr><br />
<td> BglⅡ(TOYOBO) </td><td> 1uL </td><Td> 1uL </Td></tr><tr><br />
<td> 100×BSA </td><td> 0.5uL </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 39.5uL </td><Td> 36.5uL </Td></tr><tr><br />
<td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><br><br />
<br />
3. Agarose gel electrophoresis of the digested DNA <br />
<br><br />
The digested DNA was applied to the agarose gel electrophoresis and linearized DNA was purified by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photograph of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d0/0825kit.png" width="500" height="300"><br />
<br><br><br />
4. Digestion of pSB1C3DNA with EcoRⅠ and SpeⅠ<br />
<br><br />
pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ at 37℃ for 16 hours.<br />
<br><BR><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA sample </td><td> 23uL </td></tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<tr><td> EcoRⅠ-HF(NEB) </td><td> 1uL </td></tr><tr><br />
<td> SpeⅠ(NEB) </td><td> 1.5uL </td></tr><br />
<td> 100×BSA </td><td> 0.5uL </td></tr><br />
<td> dH<sub>2</sub>O </td><td> 19uL </td></tr><br />
<td> Total <td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
<br />
5. PCR amplification of the UAS region and pSB1C3 DNA<br />
<br><br />
DNA fragments containing UAS was amplified from the pUAST DNA and the BglⅡ site-containing pSB1C3 was also amplified by PCR.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA template </td><td> 0.5uL </td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 10uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 10uL </td></tr><tr><br />
<td> 25mM MgSO4 </td><td> 3.2uL </td></tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<td> KOD plus </td><td> 2uL </td></tr><br />
<td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<td> Total <td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Circle </Td></Tr><br />
<tr><td> 95℃</td><td> 2min</td><Td></Td></tr><br />
<Tr><Td> 95℃</Td><Td> 15sec</Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃</td><td> 30min(UAS) or 2min10sec(pSB1C3)</td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃</td><td> 2min30sec</td><Td> 25 cycle </Td></tr><br />
<td> 68℃</td><td> 2min30sec</td><Td></Td></tr><br />
<td> 14℃</td><td> ∞</td><Td></Td></tr><br />
</Table><br />
<br><br><br />
<br />
<h2>August 26th</h2><br />
<br><br />
・Purification of the digested pSB1C3 <br />
<br><br />
pSB1C3 DNA double-digested with EcoRⅠ and SpeⅠ(prepared on 8/25) was applied to the agarose gel electrophoresis. The DNA fragment of interest was purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/0826.png" width="500" height="300"><br />
<br><br />
<br><br />
<h2>August 27th</h2><br />
<br><br />
1. Removing the multi-cloning site of the pEGFP-C2<br />
<br><br />
pEGFP-C2 DNA digested with BglⅡ and BamHⅠwas self ligated in the following reaction.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pEGFP-C2(cut) </Td><Td> 1uL </Td></Tr><br />
<tr><td> Ligation high </td><td> 2.5uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 5uL </td></tr><tr><br />
<td> Total <td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
2. The ligated products were transformed into the E. coli XL-1 blue and the E. coli was apreaded on the LB Kanamycin(+) plate and cultured at 37℃ for 16 hours.<br />
<br><br><br />
3. Agarose gel electrophoresis of the DNA fragments containing UAS and the PSR products from the pSB1C3 <br />
<br><br />
PCRproducts prepared on August 25th were applied to the agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/e6/0826bkit.png" width="500" height="300"><br />
<br><br><br />
Since the expected DNA fragments were detected, DNA was purified from the agarose gel by QIA quick Gel Extraction Kit. The purified DNAs were applied to the agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/99/0826ckit.png" width="500" height="300"><br />
<br><br><br />
<br />
<h2>August 28th</h2><br />
<br><br />
1. Single colony isolation from the E. coli transformed with pEGFP-C2 DNA<br />
<br><br />
Single colony isolationwas carried out from the plate prepared on 8/27and cultured in 2.5mL LB Kanamycin(+) medium at 37℃ for 16 hours.<br />
<br><br><br />
2. Transformation of E. coli with pGaTB DNA or pAct5C-GAL4 DNA<br />
<br><br />
E. coli Xl-1 blue was transformed with pGaTBDNA or pAct5C-GAL4 DNA and cultured on LB ampicillin(+) plate for 16 hours at 37℃.<br />
<br><br><br />
<br />
<h2>August 29th</h2><br />
<br><br />
1. Single colony isolation from the E. coli transformed with pGaTB DNA or pAct5C-GAL4 DNA<br />
<br><br />
From the plate prepared on 8/28 single colonies were isolated and cultured in 2.5mL LB ampicillin(+) medium for 16 hours at 37℃.<br />
<br><br><br />
2. Purification of pEGFP-C2 DNA<br />
<br><br />
pEGFP-C2 DNA was purified from E. coli prepared on 8/28 by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
3. Cut check of the pEGFP-C2 DNA<br />
<br><br />
The purified pEGFP-C2 was digested with XhoⅠ and PstⅠ in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pEGFP-C2-1, -2, -3, -4 </Td><Td> 1uL </Td></Tr><br />
<tr><td> 10×H buffer(TOYOBO) </td><td> 0.5uL </td></tr><tr><br />
<tr><td> XhoⅠ </td><td> 0.1uL </td></tr><tr><br />
<tr><td> PstⅠ </td><td> 0.1uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 3.3uL </td></tr><tr><br />
<td> Total <td> 5uL </td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were applied to the agarose gel electrophoresis.as shown below. <br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/97/0828kit.png" width="500" height="300"><br />
<br><br><br />
Results: The DNAs were undigested by these enzymes, confirming that the multi-cloning site of the pEGFP-C2 was successfully removed.<br />
<br><br><br />
4. PCR amplification of the DNA fragments containing UAS and EGFP<br />
<br><br />
Since we found that the previously used primers for PCR were wrong, PCR amplification of the DNA fragments containing UAS and EGFP region were carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 10uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 10uL </Td></Tr><br />
<Tr><Td> 25mM MgSO4 </Td><Td> 3.2uL </Td></Tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<Tr><td> KOD plus </td><td> 2uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<Tr><td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 1min(EGFP) </td><Td> 25 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 1min(EGFP) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
Agarose gel electrophoresis of the PCR products<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d2/0829b.png" width="500" height="300"><br />
<br><br><br />
Results: DNA fragments containing EGFP were successfully amplified, but not for the UAS.<br />
<br><br><br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week2p">>>>>>>>>>WEEK2</a></div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week1p
Team:KIT-Kyoto/Notebook-week1p
2012-09-26T05:16:21Z
<p>Kazuko: </p>
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<h2>August 23rd</h2><br />
<br><br />
Transformation of E. coli with pEGFP-C2 DNA and pAct5C-GAL4 DNA<br />
<br><br />
E. coli transformed with pEGFP-C2 was spread on the LB Kanamycin(+) plate and that transformed with pAct5C GAL4 was spread on the LB ampicillin(+) plate.<br />
<br />
<br><br><br />
<br />
<br />
<h2>August 24th</h2><br />
<br><br />
The appropriate colonies were picked up and cultured in the LB medium containing the appropriate antibiotics.<br />
<br><br><br />
<br />
<br />
<h2>August 25th</h2><br />
<br><br />
1. Purification of the pEGFP-C2 DNA and the pAct5C-GAL4 DNA<br />
<br><br />
These plasmid DNAs were purified from E. coli by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
2. Digestion of pEGFP-C2 DNA with BamHⅠ and BglⅡt<br />
<br><br />
Restriction enzyme digestion was carried out in the following reaction<br />
<br><br><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Total DNA amount </Td><Td> 500ng </Td><Td> 1ug </Td></Tr><br />
<tr><td> pEGFP-C </td><td> 3uL </td><Td> 6uL </Td></tr><br />
<Tr><Td> H buffer(TOYOBO) </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> BamHⅠ(TOYOBO) </td><td> 1uL </td><Td> 1uL </Td></tr><tr><br />
<td> BglⅡ(TOYOBO) </td><td> 1uL </td><Td> 1uL </Td></tr><tr><br />
<td> 100×BSA </td><td> 0.5uL </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 39.5uL </td><Td> 36.5uL </Td></tr><tr><br />
<td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><br><br />
<br />
3. Agarose gel electrophoresis of the digested DNA <br />
<br><br />
The digested DNA was applied to the agarose gel electrophoresis and linearized DNA was purified by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photograph of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d0/0825kit.png" width="500" height="300"><br />
<br><br><br />
4. Digestion of pSB1C3DNA with EcoRⅠ and SpeⅠ<br />
<br><br />
pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ at 37℃ for 16 hours.<br />
<br><BR><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA sample </td><td> 23uL </td></tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<tr><td> EcoRⅠ-HF(NEB) </td><td> 1uL </td></tr><tr><br />
<td> SpeⅠ(NEB) </td><td> 1.5uL </td></tr><br />
<td> 100×BSA </td><td> 0.5uL </td></tr><br />
<td> dH<sub>2</sub>O </td><td> 19uL </td></tr><br />
<td> Total <td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
<br />
5. PCR amplification of the UAS region and pSB1C3 DNA<br />
<br><br />
DNA fragments containing UAS was amplified from the pUAST DNA and the BglⅡ site-containing pSB1C3 was also amplified by PCR.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA template </td><td> 0.5uL </td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 10uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 10uL </td></tr><tr><br />
<td> 25mM MgSO4 </td><td> 3.2uL </td></tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<td> KOD plus </td><td> 2uL </td></tr><br />
<td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<td> Total <td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Circle </Td></Tr><br />
<tr><td> 95℃</td><td> 2min</td><Td></Td></tr><br />
<Tr><Td> 95℃</Td><Td> 15sec</Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃</td><td> 30min(UAS) or 2min10sec(pSB1C3)</td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃</td><td> 2min30sec</td><Td> 25 cycle </Td></tr><br />
<td> 68℃</td><td> 2min30sec</td><Td></Td></tr><br />
<td> 14℃</td><td> ∞</td><Td></Td></tr><br />
</Table><br />
<br><br><br />
<br />
<h2>August 26th</h2><br />
<br><br />
・Purification of the digested pSB1C3 <br />
<br><br />
pSB1C3 DNA double-digested with EcoRⅠ and SpeⅠ(prepared on 8/25) was applied to the agarose gel electrophoresis. The DNA fragment of interest was purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/0826.png" width="500" height="300"><br />
<br><br />
<br><br />
<h2>August 27th</h2><br />
<br><br />
1. Removing the multi-cloning site of the pEGFP-C2<br />
<br><br />
pEGFP-C2 DNA digested with BglⅡ and BamHⅠwas self ligated in the following reaction.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pEGFP-C2(cut) </Td><Td> 1uL </Td></Tr><br />
<tr><td> Ligation high </td><td> 2.5uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 5uL </td></tr><tr><br />
<td> Total <td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
2. The ligated products were transformed into the E. coli XL-1 blue and the E. coli was apreaded on the LB Kanamycin(+) plate and cultured at 37℃ for 16 hours.<br />
<br><br><br />
3. Agarose gel electrophoresis of the DNA fragments containing UAS and the PSR products from the pSB1C3 <br />
<br><br />
PCRproducts prepared on August 25th were applied to the agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/e6/0826bkit.png" width="500" height="300"><br />
<br><br><br />
Since the expected DNA fragments were detected, DNA was purified from the agarose gel by QIA quick Gel Extraction Kit. The purified DNAs were applied to the agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/99/0826ckit.png" width="500" height="300"><br />
<br><br><br />
<br />
<h2>August 28th</h2><br />
<br><br />
1. Single colony isolation from the E. coli transformed with pEGFP-C2 DNA<br />
<br><br />
Single colony isolationwas carried out from the plate prepared on 8/27and cultured in 2.5mL LB Kanamycin(+) medium at 37℃ for 16 hours.<br />
<br><br><br />
2. Transformation of E. coli with pGaTB DNA or pAct5C-GAL4 DNA<br />
<br><br />
E. coli Xl-1 blue was transformed with pGaTBDNA or pAct5C-GAL4 DNA and cultured on LB ampicillin(+) plate for 16 hours at 37℃.<br />
<br><br><br />
<br />
<h2>August 29th</h2><br />
<br><br />
1. Single colony isolation from the E. coli transformed with pGaTB DNA or pAct5C-GAL4 DNA<br />
<br><br />
From the plate prepared on 8/28 single colonies were isolated and cultured in 2.5mL LB ampicillin(+) medium for 16 hours at 37℃.<br />
<br><br><br />
2. Purification of pEGFP-C2 DNA<br />
<br><br />
pEGFP-C2 DNA was purified from E. coli prepared on 8/28 by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
3. Cut check of the pEGFP-C2 DNA<br />
<br><br />
The purified pEGFP-C2 was digested with XhoⅠ and PstⅠ in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pEGFP-C2-1, -2, -3, -4 </Td><Td> 1uL </Td></Tr><br />
<tr><td> 10×H buffer(TOYOBO) </td><td> 0.5uL </td></tr><tr><br />
<tr><td> XhoⅠ </td><td> 0.1uL </td></tr><tr><br />
<tr><td> PstⅠ </td><td> 0.1uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 3.3uL </td></tr><tr><br />
<td> Total <td> 5uL </td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were applied to the agarose gel electrophoresis.as shown below. <br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/97/0828kit.png" width="500" height="300"><br />
<br><br><br />
Results: The DNAs were undigested by these enzymes, confirming that the multi-cloning site of the pEGFP-C2 was successfully removed.<br />
<br><br><br />
4. PCR amplification of the DNA fragments containing UAS and EGFP<br />
<br><br />
Since we found that the previously used primers for PCR were wrong, PCR amplification of the DNA fragments containing UAS and EGFP region were carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><Td> All samples </Td></Tr><br />
<Tr><Td> DNA template </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 10uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 10uL </Td></Tr><br />
<Tr><Td> 25mM MgSO4 </Td><Td> 3.2uL </Td></Tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<Tr><td> KOD plus </td><td> 2uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<Tr><td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 1min(EGFP) </td><Td> 25 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 1min(EGFP) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
Agarose gel electrophoresis of the PCR products<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d2/0829b.png" width="500" height="300"><br />
<br><br><br />
Results: DNA fragments containing EGFP were successfully amplified, but not for the UAS.<br />
<br><br><br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week2p">>>>>>>>>>WEEK2</a></div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Project
Team:KIT-Kyoto/Project
2012-09-26T04:25:26Z
<p>Kazuko: </p>
<hr />
<div>{{KIT-Kyoto.Header}}<br />
{{KIT-Kyoto}}<br />
<br />
<html><br />
<div id="NAKAMI"><br />
<div id="Overview"><h2>Project Overview</h2><br />
<br>We try to develop new medicine for therapy of leukemia which is more free from side effects. <Br>For this study, we are going to use <I>Drosophila melanogaster</I>, a model organism to establish transgenic fly carrying responsible genes for human leukemia.<br />
</div><br />
<br><br />
<br><br />
<div id="Introduction"><h2>Introduction</h2><br />
<br><br />
<I>Drosophila melanogaster</I> has been used for a genetic study as model organism for a long time and brought us much discovery. And we are sure that the benefit continues from now on.<br />
<br><br />
Therefore we KIT-Kyoto team aim at the production of the disease model Drosophila which expresses the responsible gene of MALT lymphoma that is one of leukemia that we were not able to achieve in a meeting of the last year. It is thought that we can contribute to elucidation of the mechanism of this disease and the development of the therapeutic drug by promoting this project.<br />
<br><br />
In addition, we think about what we can do in order to continue researches using Drosophila melanogaster in the world. The structure and behavior of the gene cluster are often found by pushing forward the study about genes systematically and cooperatively.<br />
<br><br />
So, this year we aim at the design of the parts with which a study that we use the Drosophila can expand in iGEM in future.<br />
<br><br />
If these projects are realized, the study using D. melanogaster will step forward to the new one step again.<br />
</div><br />
<br><br />
<br><br />
<div id="Results"><br />
<h2>Results</h2><br />
<br><br />
<strong>BBa_K758006(UAS-EGFP)</strong><br />
<br><br><br />
We transfected this plasmid to Drosophila culture cells under two different conditions. We also transfected pAct5C-GAL4 plasmids which express GAL4 proteins in a culture cell line but not in other cell line.<br />
<br><br />
If the BBa_K758006 was assembled accurately, this plasmid expresses enough EGFP by activation of GAL4 protein. So the cell line that were co-transfected with pAct5C-GAL4 shows strong fluorescence, than the one without co-transfection with pAct5C-GAL4.<br />
<br><br><br />
Then we conducted experiment mentioned above. We made two groups of Drosophila culture cells. One group had been transfected with BBa_K758006 and pAct5C-GAL4, and the other one had been transfected with BBa_K758006 alone. And we calculated ratios between the number of green lighted cells to total cells about both groups.<br />
<br><br><br />
As results shown below, some green cells were detected among the cells co-transfected with both plasmids. But few green cells were detected among the cells transfected BBa_K758006 alone.<br />
<br><br />
And we also compiled statistics on the ratio of green lighted cells. In consequence, in the group transfected with pAct5C-GAL4, there were 45 green colored cells (12.6%) among 356 cells in total. And, without pAct5C-GAL4, there were only 2 cells were colored green (it is 0.5%) among 350 cells in total.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/f/f7/WIKI-Akit.png" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/c/c8/WIKI-Bkit.png" width="200" height="200"><br />
<br><br><br />
Cells transfected with BBa_K758006 and pAct5C-GAL4<br />
<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/6/63/WIKI-Ckit.png" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/3/31/WIKI-Dkit.png" width="200" height="200"><br />
<br><br><br />
Cells transfected with BBa_K758006 alone<br />
<br><br><br />
These results indicate that BBa_K758006 was activated by GAL4 protein and BB_K758006 EGFP expression was induced by GAL4 protein.<br />
<br><br />
In these circumstances, BBa_K758006 was working as expected.<br />
<br><br />
<br><br />
<br><br />
<br />
<br />
<br />
<strong>BBa_K758005(UAS-LacZ)</strong><br />
<br><br />
We transfected Drosophila cells with this plasmid, too. We devided the cells into two groups. One group had been co-transfected with pAct5C-GAL4 again, and the other group had been transfected with this plasmid alone. <br />
<br><br />
If BBa_K758005 was assembled accurately, the former group expresses LacZ strongly and the latter expresses weakly. <br />
<br><br><br />
Generally, to test the activation of LacZ, 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal) is used by mesuring blue color made by the degradation of X-Gal. However, in this experiment, we conducted immunostaining by red color to test the expression of LacZ (picture F, G, I, and J). Also we use 4', 6-diamidino-2-phenylindole (DAPI) that binds and emit blue light in order to count cells (picture E and H).<br />
<br><br><br />
As results shown below,some red cells were detected among the cells transfected with pAct5C-GAL4. They prove that LacZ is activated in this group. But the other group without transfection with pAct5C-GAL4 contains no red cells.<br />
<br><br />
We counted cells and calculated ratios of red cells to total cells between these two cases. In consequence, by the transfection of pAct5C-GAL4, 10.1%(42/415) cells showed red color. But, without pAct5C-GAL4, no cells showed red color (0/118 ).<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/b/bd/WIKI-Ekit.png" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/2/2d/WIKI-Fkit.png" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/2/2f/WIKI-Gkit.png" width="200" height="200"><br />
<br><br />
Drosophila cells transfected with BBa_K758005 and pAct5C-GAL4<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/5/53/WIKI-Hkit.png" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/1/16/WIKI-Ikit.png" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/6/63/WIKI-Jkit.png" width="200" height="200"><br />
<br><br />
Drosophila cells transfected with BBa_K758005 alone<br />
<br><br />
<br><br />
These results show that BBa_K758005 was activated and expression of LacZ were induced in the presence of the GAL4 protein.<br />
<br><br><br />
Therfore, it can be said that BBa_K758005 was working as expected.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<strong>Establishment of transgenic flies carrying pUAS-flag-TNFAIP3</strong><br />
<br><br><br />
We have injected 692 embryos (w-, delta2-3) with pUAS-flag-TNFAIP3 DNA (1mg/ml in microinjection buffer). Out of them 144 were hatched to larvae and 83 were further eclosed to adults. These 83 adult flies were crossed with yw flies and their progeny flies were inspected to identify successfully transformed w+ red eye flies. The red eye screening is still ongoing. However, we have identified six transformant strains that are listed below. Chromosomal linkage of the transgene is now under investigation.<br />
<br><br><br />
Strain 7, Strain 14, Strain 23, Strain 29, Strain 56, Strain 65, <br />
<br><br />
Strain 10<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/c/c1/HAEE2.JPG" width="320" height="220"><br />
<br><br />
Strain 13<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/a/a6/Strain13.JPG" width="320" height="220"><br />
<br><br />
<br />
<br />
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Kazuko
http://2012.igem.org/File:HAEE2.JPG
File:HAEE2.JPG
2012-09-26T04:24:36Z
<p>Kazuko: </p>
<hr />
<div></div>
Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-Design
Team:KIT-Kyoto/Notebook-Design
2012-09-26T04:18:30Z
<p>Kazuko: </p>
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<td width="785px" valign="top"><div id="MIGI"><br />
<h2>2012.07.03</h2><br />
<br><br />
We stared to make our logo. It needs to be more considered.<br />
<br><br><br />
<h2>2012.07.14</h2><br />
<br><br />
We made the poster for open campus.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/98/スライド2.gif" width="300" height="400"><br />
<br><br><br />
<h2>2012.08.21</h2><br />
<br><br />
We discussed our logo.<br />
<br><br />
We also talked about top images.<br />
<br><br><br />
<h2>2012.08.25</h2><br />
<br><br />
Upload our top images!<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/7/7e/KIT-KyotoA.jpg" width="280" height="50"><img src="https://static.igem.org/mediawiki/2012/0/02/KIT-KyotoB.jpg" width="280" height="50"><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9b/KIT-KyotoC.jpg" width="280" height="50"><img src="https://static.igem.org/mediawiki/2012/2/29/KIT-KyotoD.jpg" width="280" height="50"><br />
<br><br><br />
<h2>2012.08.29</h2><br />
<br><br />
The logo has decided to be adopted.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/f/ff/スライド11.jpg" width="300" height="200"><br />
<BR><br />
<h2>2012/09.03</h2><br />
<br><br />
Upload the menubar.<br />
<br><br />
<br><br />
<Img src="https://static.igem.org/mediawiki/2012/e/ee/KITIgem.jpg" width="150" height="30"><br />
<br><br />
<br><br />
<h2>2012.09.07</h2><br />
<br><br />
Upload our wiki<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/7/70/スクリーンショット_2012-09-10.png" width="450" height="300"><br />
<br><br />
<br><br />
<h2>2012.09.10</h2><br />
<br><br />
Upload sidebar.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/Side_week2kit.jpg" width="150" height="30"><br />
<br><br />
<br><br />
<h2>2012.09.18</h2><br />
<br><br />
Name cards have been completed.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/1/1d/A名刺kit.jpg" width="520" height="180"><br />
<br><br />
<br><br />
<h2>2012.09.19</h2><br />
<br><br />
Home complete.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/4/4b/スクリーンショット_2012-09-20_17.33.45.png" width="450" height="300"><br />
<br><br><br />
<h2>2012.09.21</h2><br />
<br><br />
Profile complete.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/スクリーンショット_2012-09-21_10.39.36.png" width="450" height="300"><br />
<br><br><br />
<h2>2012.09.22</h2><br />
<br><br />
Human practice complete.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/b7/スクリーンショット_2012-09-22_11.43.39.png" width="450" height="300"><br />
<h2>2012.09.24</h2><br />
<br><br />
Our Tshirt design idea has been completed!<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/1/13/KITTシャツ.png" width="450" height="300"><br />
<br><br />
</div><br />
</table><br />
</td></tr><br />
</table><br />
</body><br />
</div><br />
</html></div>
Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-september
Team:KIT-Kyoto/Notebook-Meeting-september
2012-09-26T04:18:09Z
<p>Kazuko: </p>
<hr />
<div>{{KIT-Kyoto.Header}}<br />
{{KIT-Kyoto}}<br />
<html><br />
<head><br />
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ul.menu li{<br />
padding-left:15px;<br />
margin:0;<br />
list-style: none;<br />
}<br />
div.category {<br />
margin-top: 5px;<br />
padding-left: 15px;<br />
height: 32px;<br />
line-height: 40px;<br />
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ul.menu a {<br />
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</ul><br />
</li><br />
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<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week1"><img src="https://static.igem.org/mediawiki/2012/1/1b/Side_week1kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week2"><img src="https://static.igem.org/mediawiki/2012/9/94/Side_week2kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week3"><img src="https://static.igem.org/mediawiki/2012/e/e0/Side_week3kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week4"><img src="https://static.igem.org/mediawiki/2012/3/31/Side_week4kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week5"><img src="https://static.igem.org/mediawiki/2012/e/e7/Side_week5kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week6"><img src="https://static.igem.org/mediawiki/2012/e/e8/Side_week6kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week7"><img src="https://static.igem.org/mediawiki/2012/b/b2/Side_weekkit7.jpg" width="150" height="30"></a></li><br />
</ul><br />
</li><br />
<li><br />
<div class="category"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Protocol"><img src="https://static.igem.org/mediawiki/2012/e/ee/Side_protocolkit.jpg" width="150" height="30"></a></div><br />
</li><br />
<li><br />
<div class="category"><img src="https://static.igem.org/mediawiki/2012/6/61/Side_meetingkit.jpg" width="150" height="30"></div><br />
<ul class="menu"><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-may"><img src="https://static.igem.org/mediawiki/2012/5/5d/Side_maykit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-june"><img src="https://static.igem.org/mediawiki/2012/9/92/Side_junekit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-july"><img src="https://static.igem.org/mediawiki/2012/f/f2/Side_julykit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-august"><img src="https://static.igem.org/mediawiki/2012/f/fa/Side_augkit.jpg" width="150" height="30"></a></li><li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-september"><img src="https://static.igem.org/mediawiki/2012/8/8b/Side_sepkit.jpg" width="150" height="30"></a></li><br />
</ul><br />
</li><br />
<li><br />
<div class="category"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Design"><img src="https://static.igem.org/mediawiki/2012/8/8f/Side_designnotekit.jpg" width="150" height="30"></a></div><br />
</li><br />
</ul><br />
</div><br />
<br><br />
</td><br />
<br />
<td width="800px" valign="top"><div id="MIGI"><br />
<h2>September 4th</h2><br />
<br><br />
At the library<br />
<br><br />
1. Abstract<br />
<br><br />
Our abstract is completed.<br />
<br><br />
We have to consider the title by September 7th.<br />
<br><br><br />
2. Wiki<br />
<br><br />
Some people were taken pictures to use wiki’s member page.<br />
<br><br><br />
3. Contact<br />
<br><br />
Students who are going to join Asia Jamboree have to register by September 7.<br />
<br><br><br />
<br />
<h2>September 13th</h2><br />
<br><br />
At the library<br />
<br><br />
1. Design<br />
<br><br />
We almost completed the designs of the business card and T-shirts.<br />
<br><br />
Boo has ordered T-shirts.<br />
<br><br><br />
2. Release form<br />
<br><br />
Students who are going to join Asia Jamboree have to fill in the blanks of release form by Asia Jamboree and take it.<br />
<br><br><br />
3. Homework and Contact<br />
<br><br />
Tamiaki and Sakamoto must check the past presentation and poster by September 18th. They should make time for iGEM next week.<br />
<br><br />
</div><br />
</div><br />
</table><br />
</td></tr><br />
<br />
</table><br />
</body><br />
<br />
<br />
<br />
</html></div>
Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-august
Team:KIT-Kyoto/Notebook-Meeting-august
2012-09-26T04:17:38Z
<p>Kazuko: </p>
<hr />
<div>{{KIT-Kyoto.Header}}<br />
{{KIT-Kyoto}}<br />
<html><br />
<head><br />
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<script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.4.2/jquery.min.js"></script><br />
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div.category {<br />
margin:-top:5px;<br />
height:40px;<br />
line-height:40px;<br />
cursor:pointer;<br />
}<br />
ul.navi{<br />
width: 150px;<br />
margin: 0px;<br />
}<br />
ul.navi, ul.menu {<br />
padding:0;<br />
margin:0;<br />
list-style: none;<br />
}<br />
ul.menu li{<br />
padding-left:15px;<br />
margin:0;<br />
list-style: none;<br />
}<br />
div.category {<br />
margin-top: 5px;<br />
padding-left: 15px;<br />
height: 32px;<br />
line-height: 40px;<br />
}<br />
ul.menu a {<br />
display: block;<br />
height: 32px;<br />
line-height: 10px;<br />
}<br />
</style><br />
</head><br />
<body><br />
<table border="0" width="965px" align="center"><tr><td width="165px" valign="top" <br />
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align="left"><div id="HIDARI"><br />
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</ul><br />
</li><br />
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<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week2"><img src="https://static.igem.org/mediawiki/2012/9/94/Side_week2kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week3"><img src="https://static.igem.org/mediawiki/2012/e/e0/Side_week3kit.jpg" width="150" height="30"></a></li><br />
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<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week6"><img src="https://static.igem.org/mediawiki/2012/e/e8/Side_week6kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week7"><img src="https://static.igem.org/mediawiki/2012/b/b2/Side_weekkit7.jpg" width="150" height="30"></a></li><br />
</ul><br />
</li><br />
<li><br />
<div class="category"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Protocol"><img src="https://static.igem.org/mediawiki/2012/e/ee/Side_protocolkit.jpg" width="150" height="30"></a></div><br />
</li><br />
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<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-june"><img src="https://static.igem.org/mediawiki/2012/9/92/Side_junekit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-july"><img src="https://static.igem.org/mediawiki/2012/f/f2/Side_julykit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-august"><img src="https://static.igem.org/mediawiki/2012/f/fa/Side_augkit.jpg" width="150" height="30"></a></li><li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-september"><img src="https://static.igem.org/mediawiki/2012/8/8b/Side_sepkit.jpg" width="150" height="30"></a></li><br />
</ul><br />
</li><br />
<li><br />
<div class="category"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Design"><img src="https://static.igem.org/mediawiki/2012/8/8f/Side_designnotekit.jpg" width="150" height="30"></a></div><br />
</li><br />
</ul><br />
</div><br />
<br><br />
</td><br />
<br />
<td width="800px" valign="top"><div id="MIGI"><br />
<br><br />
<br />
<h2>August 7th</h2><br />
<br><br />
At the library<br />
<br><br />
1. Wiki<br />
<br><br />
When the lab note is uploaded into Dropbox, we have to translate into English (We don’t decide who should translate it.)<br />
<br><br><br />
<br />
2. Logo<br />
<br><br />
We need more discussion!<br />
<br><br><br />
<br />
3. Movie<br />
<br><br />
B1 students must prepare a draft.<br />
<br><br><br />
4. Questionnire<br />
<br><br />
We should collaborate with other universities.<br />
<br><br><br />
By Megumi<br />
<br><br><br />
<br />
<h2>August 20th</h2><br />
<br><br />
At the library<br />
<br><br />
1.We allotted a part to each member.<br />
<br><br />
Wiki Kazuko, Kouji and Yu.H.<br />
<br><br />
(Translate Meeting, Protocol and lab note into English.)<br />
<br><br />
Poster Yuichi, Megumi and Ginga<br />
<br><br />
(See 2010, 2011 best posters!)<br />
<br><br />
Presentation Yohei, Masahiro and Yuki<br />
<br><br />
(introduction from 5 to 10 pages)<br />
<br><br />
All works have to be reported on August 29th.<br />
<br><br><br />
<br />
2.Design<br />
<br><br />
We’ll make our team T-shirt. Art-course students will help make poster and powerpoint.<br />
<br><br><br />
By Kazuko<br />
<br><br><br />
<h2>August 29th</h2><br />
<br><br />
At the library<br />
<br><br />
1. Presentation by B1 students<br />
<br><br />
About poster by Ginga and Megumi<br />
<br><br />
About KIT by Masahiro<br />
<br><br />
About movie by Yohei<br />
<br><br><br />
<br />
2. Wiki<br />
<br><br />
We decided our logo!<br />
<br><br />
We have to translate the documents into English until each deadline.<br />
<br><br><br />
3.Abstract<br />
<br><br />
We have to consider the title when the abstract is completed.<br />
<br><br><br />
By Megumi<br />
<br><br />
<br><br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-september">>>>>>>>>>SEPTEMBER</a></div><br />
</div><br />
</div><br />
</table><br />
</td></tr><br />
<br />
</table><br />
</body><br />
<br />
<br />
<br />
</html></div>
Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-july
Team:KIT-Kyoto/Notebook-Meeting-july
2012-09-26T04:17:15Z
<p>Kazuko: </p>
<hr />
<div>{{KIT-Kyoto.Header}}<br />
{{KIT-Kyoto}}<br />
<html><br />
<head><br />
<meta charset="UTF-8"><br />
<script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.4.2/jquery.min.js"></script><br />
<script type="text/javascript"><br />
$(function(){<br />
$("ul.menu").hide(); <br />
$("div.category").click(function(){ <br />
$("ul.menu").slideUp();<br />
if($("+ul",this).css("display")=="none"){<br />
$("+ul",this).slideDown();<br />
}<br />
});<br />
});<br />
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<style type="text/css"><br />
body {<br />
font-family: Myriad, Helvetica, Arial, "Meiryo", "メイリオ", sans-serif;<br />
_font-family: 'MS Pゴシック', sans-serif;<br />
}<br />
div.category {<br />
margin:-top:5px;<br />
height:40px;<br />
line-height:40px;<br />
cursor:pointer;<br />
}<br />
ul.navi{<br />
width: 150px;<br />
margin: 0px;<br />
}<br />
ul.navi, ul.menu {<br />
padding:0;<br />
margin:0;<br />
list-style: none;<br />
}<br />
ul.menu li{<br />
padding-left:15px;<br />
margin:0;<br />
list-style: none;<br />
}<br />
div.category {<br />
margin-top: 5px;<br />
padding-left: 15px;<br />
height: 32px;<br />
line-height: 40px;<br />
}<br />
ul.menu a {<br />
display: block;<br />
height: 32px;<br />
line-height: 10px;<br />
}<br />
</style><br />
</head><br />
<body><br />
<table border="0" width="965px" align="center"><tr><td width="165px" valign="top" <br />
<br />
align="left"><div id="HIDARI"><br />
<br />
<br><br />
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<ul class="navi"><br />
<li><br />
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<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week2p"><img src="https://static.igem.org/mediawiki/2012/9/94/Side_week2kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week3p"><img src="https://static.igem.org/mediawiki/2012/e/e0/Side_week3kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week4p"><img src="https://static.igem.org/mediawiki/2012/3/31/Side_week4kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week5p"><img src="https://static.igem.org/mediawiki/2012/e/e7/Side_week5kit.jpg" width="150" height="30"></a></li><br />
</ul><br />
</li><br />
<li><br />
<li><br />
<div class="category"><img src="https://static.igem.org/mediawiki/2012/e/eb/Side_tnfaip3kit.jpg" width="150" height="30"></div><br />
<ul class="menu"><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week1"><img src="https://static.igem.org/mediawiki/2012/1/1b/Side_week1kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week2"><img src="https://static.igem.org/mediawiki/2012/9/94/Side_week2kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week3"><img src="https://static.igem.org/mediawiki/2012/e/e0/Side_week3kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week4"><img src="https://static.igem.org/mediawiki/2012/3/31/Side_week4kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week5"><img src="https://static.igem.org/mediawiki/2012/e/e7/Side_week5kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week6"><img src="https://static.igem.org/mediawiki/2012/e/e8/Side_week6kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week7"><img src="https://static.igem.org/mediawiki/2012/b/b2/Side_weekkit7.jpg" width="150" height="30"></a></li><br />
</ul><br />
</li><br />
<li><br />
<div class="category"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Protocol"><img src="https://static.igem.org/mediawiki/2012/e/ee/Side_protocolkit.jpg" width="150" height="30"></a></div><br />
</li><br />
<li><br />
<div class="category"><img src="https://static.igem.org/mediawiki/2012/6/61/Side_meetingkit.jpg" width="150" height="30"></div><br />
<ul class="menu"><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-may"><img src="https://static.igem.org/mediawiki/2012/5/5d/Side_maykit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-june"><img src="https://static.igem.org/mediawiki/2012/9/92/Side_junekit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-july"><img src="https://static.igem.org/mediawiki/2012/f/f2/Side_julykit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-august"><img src="https://static.igem.org/mediawiki/2012/f/fa/Side_augkit.jpg" width="150" height="30"></a></li><li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-september"><img src="https://static.igem.org/mediawiki/2012/8/8b/Side_sepkit.jpg" width="150" height="30"></a></li><br />
</ul><br />
</li><br />
<li><br />
<div class="category"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Design"><img src="https://static.igem.org/mediawiki/2012/8/8f/Side_designnotekit.jpg" width="150" height="30"></a></div><br />
</li><br />
</ul><br />
</div><br />
<br><br />
</td><br />
<br />
<td width="800px" valign="top"><div id="MIGI"><br />
<br><br />
<br />
<h2>July 3rd</h2><br />
<br><br />
At the library<br />
<br><br />
1.Logo<br />
<br><br />
We talked about our logo. Its image is like a classical Japanese family crest. <br />
<br><br />
Art-course students made the logo of a different version (overhead-view version of Drosophila) from that on July 10th.<br />
<br><br><br />
2.Questionnaire<br />
<br><br />
We corrected mistakes of the questionnaire. We have to find people who are likely to fill out our questionnaire (especially at the offices).<br />
<br><br><br />
3.Schedule<br />
<br><br />
On July 13th : the last meeting before periodical exams<br />
<br><br />
On July 15th : deadline of Description<br />
<br><br />
Oct 5th~Oct 8th : Asia Jamboree<br />
<br><br><br />
4.Photos<br />
<br><br />
We decided to take photos related with iGEM and send to Dropbox.<br />
<br><br><br />
By Kazuko<br />
<br><br><br />
<h2>July 6th</h2><br />
<br><br />
In classroom 0221 of the 2nd Bldg.<br />
<br><br />
1. Questionnaire<br />
<br><br />
We finally completed our questionnaire! <br />
<br><br />
When we distribute this, we can change only its introduction according to personal matters.<br />
<br><br><br />
2. Wiki<br />
<br><br />
In order to decide the layout, each of us has to look back past wiki pages. <br />
<br><br />
We’ll discuss more in detail with art-course students next Tuesday.<br />
<br><br><br />
3. Open campus<br />
<br><br />
We almost completed the panel for KIT open campus.<br />
<br><br><br />
4. Contact<br />
<br><br />
Students who are going to join Asia Jamboree have to mail to Shunji.<br />
<br><br><br />
5.Twitter<br />
<br><br />
We got a new twitter account of KIT-Kyoto12 ! (igem2012kitkyoto@aol.jp)<br />
<br><br><br />
By Megumi<br />
<br><br><br />
<h2>July 10th</h2><br />
<br><br />
At the library<br />
<br><br />
1. Logo<br />
<br><br />
We talked about our logo. <br />
<br><br><br />
<br />
2. Wiki<br />
<br><br />
Its image is Japanese style. Our image color is light purple and pink. Art-course students have to make the top images.<br />
<br><br><br />
By Kazuko<br />
<br><br><br />
<h2>July 13th</h2><br />
<br><br />
At the library<br />
<br><br />
We talked about logo and wiki.<br />
<br><br />
Today's meeting is the last one in the first semester. Good luck!<br />
<br><br><br />
By Megumi<br />
<br><br />
<br><br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-august">>>>>>>>>>AUGUST</a></div><br />
</div><br />
</div><br />
</table><br />
</td></tr><br />
<br />
</table><br />
</body><br />
<br />
<br />
<br />
</html></div>
Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-june
Team:KIT-Kyoto/Notebook-Meeting-june
2012-09-26T04:16:47Z
<p>Kazuko: </p>
<hr />
<div>{{KIT-Kyoto.Header}}<br />
{{KIT-Kyoto}}<br />
<html><br />
<head><br />
<meta charset="UTF-8"><br />
<script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.4.2/jquery.min.js"></script><br />
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<table border="0" width="965px" align="center"><tr><td width="165px" valign="top" <br />
<br />
align="left"><div id="HIDARI"><br />
<br />
<br><br />
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<ul class="navi"><br />
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<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week1p"><img src="https://static.igem.org/mediawiki/2012/1/1b/Side_week1kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week2p"><img src="https://static.igem.org/mediawiki/2012/9/94/Side_week2kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week3p"><img src="https://static.igem.org/mediawiki/2012/e/e0/Side_week3kit.jpg" width="150" height="30"></a></li><br />
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<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week5p"><img src="https://static.igem.org/mediawiki/2012/e/e7/Side_week5kit.jpg" width="150" height="30"></a></li><br />
</ul><br />
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<div class="category"><img src="https://static.igem.org/mediawiki/2012/e/eb/Side_tnfaip3kit.jpg" width="150" height="30"></div><br />
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<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week1"><img src="https://static.igem.org/mediawiki/2012/1/1b/Side_week1kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week2"><img src="https://static.igem.org/mediawiki/2012/9/94/Side_week2kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week3"><img src="https://static.igem.org/mediawiki/2012/e/e0/Side_week3kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week4"><img src="https://static.igem.org/mediawiki/2012/3/31/Side_week4kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week5"><img src="https://static.igem.org/mediawiki/2012/e/e7/Side_week5kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week6"><img src="https://static.igem.org/mediawiki/2012/e/e8/Side_week6kit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week7"><img src="https://static.igem.org/mediawiki/2012/b/b2/Side_weekkit7.jpg" width="150" height="30"></a></li><br />
</ul><br />
</li><br />
<li><br />
<div class="category"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Protocol"><img src="https://static.igem.org/mediawiki/2012/e/ee/Side_protocolkit.jpg" width="150" height="30"></a></div><br />
</li><br />
<li><br />
<div class="category"><img src="https://static.igem.org/mediawiki/2012/6/61/Side_meetingkit.jpg" width="150" height="30"></div><br />
<ul class="menu"><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-may"><img src="https://static.igem.org/mediawiki/2012/5/5d/Side_maykit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-june"><img src="https://static.igem.org/mediawiki/2012/9/92/Side_junekit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-july"><img src="https://static.igem.org/mediawiki/2012/f/f2/Side_julykit.jpg" width="150" height="30"></a></li><br />
<li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-august"><img src="https://static.igem.org/mediawiki/2012/f/fa/Side_augkit.jpg" width="150" height="30"></a></li><li><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-september"><img src="https://static.igem.org/mediawiki/2012/8/8b/Side_sepkit.jpg" width="150" height="30"></a></li><br />
</ul><br />
</li><br />
<li><br />
<div class="category"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Design"><img src="https://static.igem.org/mediawiki/2012/8/8f/Side_designnotekit.jpg" width="150" height="30"></a></div><br />
</li><br />
</ul><br />
</div><br />
<br><br />
</td><br />
<br />
<td width="800px" valign="top"><div id="MIGI"><br />
<h2>June 5th</h2><br />
<br><br />
At Building 2, Room 0223<br />
<br><br />
1. Description<br />
<br><br />
Title should be 2 or 3 sentence long and contains less than 10 words.<br />
<br><br />
Write after the researches of pharmaceutical.<br />
<br><br />
2. Presentation<br />
<br><br />
Give PowerPoint for modeling information including pharmaceutical.<br />
<br><br />
Study about side effects of main pharmaceuticals.<br />
<br><br />
3. Open Campus<br />
<br><br />
Hold on 8/10,11<br />
<br><br />
Prepare panels and materials for explanation<br />
<br><br />
4. Sponsor<br />
<br><br />
Send documents by a handwriting.<br />
<br><br />
Ask participants of last year for some advice.<br />
<br><br><br />
Edited by Yohei<br />
<br><br><br />
<h2>June 8th</h2><br />
<br><br />
At Building 2, Room 0223<br />
<br><br />
about Human practice<br />
<br><br />
Try to contribute to a video site<br />
<br><br />
Planning of the setup<br />
<br><br />
Important note, Actual movie, Staffroll like promotion videos<br />
<br><br />
In order to show that synthetic biology is safe. And ask not only impression of genetic engineering but also reasons of their answers to our questire.<br />
<br><br><br />
Edited by Megumi<br />
<br><br><br />
<h2>June 12th</h2><br />
<br><br />
At Bld 2, Rm 0223<br />
<br><br />
1. Open Campus<br />
<br><br />
2. Description<br />
<br><br />
Make sure of current medicines for leukemia<br />
<br><br />
Write articles in Japanese (Watanabe, Okumura, Murakami)<br />
<br><br><br />
Edited by Yuichi<br />
<br><br><br />
<h2>June 15th</h2><br />
<br><br />
At Bld 2, Rm 0223<br />
<br><br />
1. Ask modeling staffs to make project Logo(Japanese style, including a fly picture)<br />
<br><br />
2. Description<br />
<br><br />
Finish writing articles in Japanese<br />
<br><br />
私たちは副作用の少ない白血病の新薬の作成を試みます。そのため、ヒトの白血病の原因遺伝子を組み込んだモデ ル生物であるショウジョウバエの利用を予定しています。<br />
<br><br />
Kazuko translates into English<br />
<br><br><br />
Edited by Kazuko<br />
<br><br><br />
<h2>June 22nd</h2><br />
<br><br />
At Laboratory 3F of library<br />
<br><br />
1. Description<br />
<br><br />
English version<br />
<br><br />
We try to develop new medicine for therapy of leukemia which is free from side effects. For this study, we are going to use Drosophila melanogaster, a model organism to establish transgenic fly carrying responsible genes for human leukemia.<br />
<br><br />
2. Presentation for modeling staffs<br />
<br><br />
What is leukemia?<br />
<br><br />
About our experimentation.<br />
<br><br />
3. Ask modeling staffs to make Logo and business card<br />
<br><br />
4. Questionnaire<br />
<br><br />
If having something to point out, mail to Hatano.<br />
<br><br><br />
<br />
<h2>June 26th</h2><br />
<br><br />
At Building 1, Room 0112<br />
<br><br />
1. Introduction for questionnaire<br />
<br><br />
2. Video Movie<br />
<br><br />
Choose casts.<br />
<br><br />
3. Open Campus<br />
<br><br />
Begin to write text.<br />
<br><br />
4. Safety<br />
<br><br />
Translate into English (Murakami, Yamauchi)<br />
<br><br><br />
<br />
Edited by Kazuko<br />
<br><br><br />
<h2>June 29th</h2><br />
<br><br />
At LaboratryB 3F of library<br />
<br />
<br><br />
1. Questionnaire<br />
<br><br />
Change a part of introduction.<br />
<br><br />
2. Video Movie<br />
<br><br />
Make a video in summer vacation.<br />
<br><br />
Continue to discuss about contents and casts.<br />
<br><br />
3. Open Campus<br />
<br><br />
Make panels by 7/29.<br />
<br><br />
4. Wiki page<br />
<br><br />
Add a page about photo gallery.<br />
<br><br><br />
Edited by Yohei<br />
<br><br><br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-july">>>>>>>>>>JULY</a></div><br />
</div><br />
</div><br />
</table><br />
</td></tr><br />
<br />
</table><br />
</body><br />
<br />
<br />
<br />
</html></div>
Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-may
Team:KIT-Kyoto/Notebook-Meeting-may
2012-09-26T04:16:22Z
<p>Kazuko: </p>
<hr />
<div>{{KIT-Kyoto.Header}}<br />
{{KIT-Kyoto}}<br />
<html><br />
<head><br />
<meta charset="UTF-8"><br />
<script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.4.2/jquery.min.js"></script><br />
<script type="text/javascript"><br />
$(function(){<br />
$("ul.menu").hide(); <br />
$("div.category").click(function(){ <br />
$("ul.menu").slideUp();<br />
if($("+ul",this).css("display")=="none"){<br />
$("+ul",this).slideDown();<br />
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margin:-top:5px;<br />
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height: 32px;<br />
line-height: 40px;<br />
}<br />
ul.menu a {<br />
display: block;<br />
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line-height: 10px;<br />
}<br />
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align="left"><div id="HIDARI"><br />
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</ul><br />
</li><br />
<li><br />
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<br><br />
</td><br />
<br />
<td width="800px" valign="top"><div id="MIGI"><br />
<h2>May 1st</h2><br />
<br><br />
At No.2 building, 2nd floor, room 223<br />
<br><br />
1. Self-introduction<br />
<br><br><br />
2. Study about safety<br />
<br><br />
HOMEWORK: We will talk about safety and human practice next week.<br />
<br><br> <br />
<h2>May 8th</h2><br />
<br><br />
At No.2 building, 2nd floor, room 223<br />
<br><br />
<br><br />
Hatano stood as a candidate for the leader of a first-year students<br />
<br><br />
Matuzaki decided to do finances<br />
<br><br />
We studied PCR and a general outline of experiments<br />
<br><br />
We started study of Drosophila and microorganism<br />
<br><br />
We must think about the date of study and progress of meeting<br />
<br><br><br />
<h2>May 11th</h2><br />
<br><br />
At No.2 building, 2nd floor, room 223<br />
<br><br />
We decided to read「Molecular Biology of the Cell」Chapter22 for study of Drosophila<br />
<br><br><br />
<h2>May 18th</h2><br />
<br><br />
At Meeting room of the department of applied biology<br />
<br><br />
We read「Molecular Biology of the Cell」<br />
<br><br />
HOMEWORK: We must decide the contents of human practice<br />
<br><br />
<br><br />
<h2>May 25th</h2><br />
<br><br />
At No.2 building, 2nd floor, room 223<br />
<br><br />
1. We read「Molecular Biology of the Cell」p.1333~p.1337<br />
<br><br><br />
2. We announced the idea of human practice<br />
<br><br />
(Ideas)<br />
<br><br />
We make HP, and we print this HP on SNS or twitter.<br />
<br><br />
We introduce iGEM to other universities at the open campus.<br />
<br><br />
We make a newspaper of iGEM.<br />
<br><br />
We notify our activity using poster or handbill.<br />
<br><br />
We explain iGME to high school student.<br />
<br><br />
We do more publicity.<br />
<br><br />
We make animation of our working.<br />
<br><br />
We make iGEM HP in Japanese with other universities joining iGEM.<br />
<br><br><br />
3. We decided the charge of each person.<br />
<br><br />
Open campus-Sakamoto(L),Watanabe,Murakami,Okumura<br />
<br><br />
A sponsor-Tanaka(L),Matuzaki,Sano,Tamiaki<br />
<br><br />
Information of Tokyo-unsettled<br />
<br><br />
Questionnaire-Hatano(L),Nishiwaki,Yamauchi<br />
<br><br />
*(L) is team leader<br />
<br><br />
Wiki-Takeda(L),Okumura,Murakami,Yamauchi<br />
<br><br><br />
4. About safety and description<br />
<br><br />
We studied about safety and description (Japanese) by June 5<br />
<br><br />
We studied about safety and description (English) by June 15<br />
<br><br />
HOMEWORK: We should read safety guideline on iGEM page.<br />
<br><br><br />
<br />
<h2>May 29th</h2><br />
<br><br />
At Meeting room of the department of applied biology (4th floor)<br />
<br><br />
1. Emily Rei Worung and Boo Kwidae join team.<br />
<br><br />
They decided to make design of card<br />
<br><br><br />
2. About safety<br />
<br><br />
Safety quiz<br />
<br><br />
Make bio-safety check list (key of the stock of chemicals and tools)<br />
<br><br><br />
3. About description<br />
<br><br />
If we understand genes of leukemia, what can we do ? <br />
<br><br />
→ medicine manufacture<br />
<br><br><br />
4. A presentation for Emily Rei Worung and Boo Kwidae.<br />
<br><br />
Drosophila-Azuma<br />
<br><br />
Leukemia-Sano Sakamoto Tamiaki<br />
<br><br />
Contents of experiments-Takeda<br />
<br><br />
Make PowerPoint by June 12<br />
<br><br />
A deadline<br />
<br><br />
Safety and Description(Japanese)-June 5<br />
<br><br />
PowerPoint-June 12<br />
<br><br />
Contents of Questionnaire-June 15<br />
<br><br />
Safety and Description(English)-June 15<br />
<br><br><br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-Meeting-june">>>>>>>>>>JUNE</a></div><br />
</div><br />
</div><br />
</table><br />
</td></tr><br />
<br />
</table><br />
</body><br />
<br />
<br />
<br />
</html></div>
Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-Protocol
Team:KIT-Kyoto/Notebook-Protocol
2012-09-26T04:15:35Z
<p>Kazuko: </p>
<hr />
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</td><br />
<br />
<td width="800px" valign="top"><div id="MIGI"><br />
<h2>LB medium</h2><br />
<br><br />
<br><br />
*per kilogram<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Bacto-tryptone </Td><Td> 10g </Td></Tr><br />
<Tr><Td> Bacto-yeast extract </Td><Td> 5g </Td></Tr><br />
<Tr><Td> NaCl </Td><Td> 10g </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 1000mL </Td></Tr><br />
</Table><br />
<br><br />
1. Mix the reagents according to the previous components. <br />
<br><br />
2. Adjust the pH (pH7.0) with NaOH. <br />
<br><br />
3. Autoclave at 120℃ for 20min<br />
<br><br />
<br><br />
<br />
<br />
<br />
<h2>LB plate</h2><br />
<br><br />
<br><br />
*per liter<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Bacto-tryptone </Td><Td> 10g </Td></Tr><br />
<Tr><Td> Bacto-yeast extract </Td><Td> 5g </Td></Tr><br />
<Tr><Td> NaCl </Td><Td> 10g </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 1000mL </Td></Tr><br />
</Table><br />
<br><br />
1. Mix the reagent according to the previous components. <br />
<br><br />
2. Add NaOH and carry the enzymes pH7.0. <br />
<br><br />
3. Add 15g Bacto agar and dissolve.<br />
<br><br />
4. Autoclave at 120℃ 20min.<br />
<br><br />
5. After cooling down approx. 60℃, add the suitable amounts of appropriate antibiotics.<br />
<br><br />
6. Pour 20~30mL into laboratory dishes.<br />
<br><br />
<br><br />
*Solution Ⅰ<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Glucose </Td><Td> 50mM </Td></Tr><br />
<Tr><Td> EDTA </Td><Td> 10mM </Td></Tr><br />
<Tr><Td> Tris-HCl </Td><Td> 25mM </Td></Tr><br />
</Table><br />
<br><br />
<br><br />
*Solution Ⅱ<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> NaOH </Td><Td> 0.2N </Td></Tr><br />
<Tr><Td> SDS </Td><Td> 1% </Td></Tr><br />
</Table><br />
<br><br />
<br><br />
*Solution Ⅲ<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> K-acetate </Td><Td> 5M </Td></Tr><br />
</Table><br />
<br><br />
Methods<br />
<br><br />
<br><br />
<strong>Transformation into plasmid of E.coli.</strong><br />
<br><br />
<br><br />
1. Dissolve competent cell on ice.<br />
<br><br />
2. Pour 100uL competent cell into 1.5mL tube, and mix with 1~5uL DNA.<br />
<br><br />
3. Incubate for 30 minutes on ice.<br />
<br><br />
4. Treat with heat shock the cells at 42℃ for 45 seconds.<br />
<br><br />
5. Cool down on ice for 2 minutes.<br />
<br><br />
6. Add 250uL S.O.C medium at room temperature.<br />
<br><br />
7. Incubate at 37℃ with shaking for 1 hour.<br />
<br><br />
8. Spread a suitable amount of the cells on LB plate (containing appropriate antibiotics).<br />
<br><br />
9. Incubate overnight at 37℃.<br />
<br><br />
<br><br />
<strong>Picking up a single colony</strong><br />
<br><br><br />
Pick up the colony grown on the LB plate by a platinum stick and put in 2.5mL of LB medium (containing appropriate antibiotics), and cultivate cells at 37˚C.<br />
<br><br />
<br><br />
<h2>Miniprep by Alkaline-SDS method.</h2><br />
<br><br />
<br><br />
1. Spin 1.5 mL of culture in microcentrifuge tube at 12,000 rpm for 5 min. at 4 ˚C. Discard the supernatant <br />
<br><br />
and spin again for 2 min. Remove remaining liquid carefully.<br />
<br><br />
2. Resuspend the bacterial cell pellet in 100 μL of solution I previously cooled on ice.<br />
<br><br />
3. After 4 min. at room temperature, add 200 μL of solution II and mix by inverting several times.<br />
<br><br />
4. Keep on ice for 4 min.<br />
<br><br />
6. Add 160 μL of solution III and mix gently by inverting several times. Keep on ice for 5 min.<br />
<br><br />
7. Centrifuge at 12,000 rpm for 10 min. at 4˚C<br />
<br><br />
8. Transfer supernatant to new tube.<br />
<br><br />
9. Add the same volume of phenol/chloroform and vortex well. Centrifuge at 12,000 rpm for 2 min at room <br />
<br><br />
temperature.<br />
<br><br />
10. Transfer top clear layer to new tube. Add the same volume of diethyl ether and vortex. Centrifuge at <br />
<br><br />
12,000 rpm for 2 min. at room temperature.<br />
<br><br />
11. Transfer the top clear layer to new tube. Add 2X volume of ethanol and vortex. Centrifuge at 12,000 rpm <br />
<br><br />
for 10 min. at room temperature.<br />
<br><br />
12. Remove supernatant and add 1 mL of 70% ethanol and mix gently. Centrifuge at 12,000 rpm for 5 min at <br />
<br><br />
room temperature.<br />
<br><br />
13. Remove supernatant and dry in vacuo. Dissolve pellet in 20 μL TE.<br />
<br><br><br />
<h2>Miniprep by QIA prep Spin Miniprep Kit</h2><br />
<br><br><br />
1. Spin 1.5 mL of culture in microcentrifuge tube at 10,000 rpm for 5 min. at 4˚C. Discard the supernatant <br />
<br><br />
and spin again for 2 min. Remove remaining liquid carefully.<br />
<br><br />
2. Resuspend the bacterial cell pellet in 250 μL of buffr P1.<br />
<br><br />
3. Keep at room temperature for 4 min.<br />
<br><br />
4. Add 250 μL of buffer P2 and mix by inverting quickly.<br />
<br><br />
5. Keep on ice for 4 min.<br />
<br><br />
6. Centrifuge at 13,000 rpm for 10 min. at 4˚C.<br />
<br><br />
7. Apply supernatant to QIAprepspin column.<br />
<br><br />
8. Centrifuge at 10,000 rpm for 40 sec. at room temperature. Discard the flow-through.<br />
<br><br />
9. Add 500 μL of buffer PB. Centrifuge at 10,000 rpm for 40 sec. at room temperature. Discard the flow-<br />
<br><br />
through.<br />
<br><br />
10. Add 750 μL of buffer PE. Centrifuge at 10,000 rpm for 40 sec. at room temperature. Discard the flow-<br />
<br><br />
through.<br />
<br><br />
11. Centrifuge again at 10,000 rpm for 1 min. at room temperature. Set a column on new 1.5 mL tube.<br />
<br><br />
12. Add 30 μL of buffer EB and keep at room temperature for 1 min. Centrifuge at 10,000 rpm for 1 min at <br />
<br><br />
room temperature.<br />
<br><br />
13. Recover purified DNA in 1.5 mL tube.<br />
<br><br />
<br><br />
<h2>Midiprep by Pure Link<sup>TM</sup> HiPure Midiprep Kit</h2><br />
<br><br />
<br><br />
1. Centrifuge (4000xg,4°C,10min) E. coli culture in LB medium (50 mL) and remove the supernatant<br />
<br><br />
2. Mix with suspension buffer (4mL) including RNase (20 micro g/mL) and the pellet <br />
<br><br />
3. Add Lysis buffer (4mL) and turn upside and down several times in order to mix well<br />
<br><br />
4. Incubate at room temperature for 5min<br />
<br><br />
5. Add precipitation buffer (4mL) and shake the tube quickly<br />
<br><br />
6. Centrifuge and remove the supernatant (15000 x g, room temperature, 10 min)<br />
<br><br />
7. Transfer the supernatant to balanced column and elute out the solution by gravity<br />
<br><br />
8. Wash the column twice with Wash buffer (10 mL) and elute out the solution as gravitation every after <br />
<br><br />
washing.<br />
<br><br />
9. Put sterilized 15 mL centrifuge tube under the column<br />
<br><br />
10. Add Elution buffer (5 mL) to the column and elute out the solution by gravity<br />
<br><br />
11. Add isopropanol (3.5 mL) to the centrifuge tube and mix well<br />
<br><br />
12. Centrifuge and remove the supernatant (15000 x g, 4°C, 30min)<br />
<br><br />
13. Add 70% ethanol and mix well<br />
<br><br />
14. Centrifuge and remove the supernatant (15000 x g, 4°C, 5min)<br />
<br><br />
15. Dehydrated ,then suspend purified DNA into TE buffer (200 uL)<br />
<br><br><br />
<h2>Purification of DNA from gel by QIA quick Gel Extraction Kit</h2><br />
<br><br />
<br><br />
1. Cut out the DNA fragment from agarose gel and put it in a 1.5mL tube and weigh the gel<br />
<br><br />
2 .Add Buffer QG (x 3 volume of the gel weight)<br />
<br><br />
3 .Vortex every 2 - 3 minutes to dissolve the gel completely <br />
<br><br />
4 . Add isopropanol (equal weight to the gel, 100uL per 100mg)<br />
<br><br />
5 . Trabsfer to spin column<br />
<br><br />
6 . Centrifuge (10000rpm, room temperature,1min)<br />
<br><br />
7 . Remove the solution<br />
<br><br />
8 . Add Buffer PE (750 uL) and centrifuge (10000rpm,room temperature,1min)<br />
<br><br />
9 . Remove the solution and centrifuge (17900 x g, room temperature, 1min)<br />
<br><br />
10. Set a new 1.5 tube under the column, add TE (30uL) and centrifuge (10000 rpm, room temperature,1min)<br />
<br><br />
11. Collect the purified DNA <br />
<BR><br><br />
<br />
<h2>BP reaction by Invitrogen gateway system</h2><br />
<br><br><br />
1. PCR using primers containing the attB sequence.<br />
<br><br />
2. Purify PCR product.<br />
<br><br />
3. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> attB PCR product </Td><Td> 75 ng/reaction (1-7 μL) </Td></Tr><br />
<Tr><Td> pDONR vector </Td><Td> 150ng/reaction (1 μL) </Td></Tr><br />
<Tr><Td> TE Buffer </Td><Td> to 8 μL </Td></Tr><br />
</Table><br />
<br><br />
4. Vortex BP ClonaseII enzyme mix briefly. Add 1 - 2 μL to the components above and mix well by vortexing<br />
<br><br />
and spin them down.<br />
<br><br />
5. Incubate reaction at 25˚C for more than 1 hour.<br />
<br><br />
6. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.<br />
<br><br />
7. Incubate at 37˚C for 10 minutes.<br />
<br><br />
<br><br />
<br />
<br />
<h2>LR reaction by Invitrogen gateway system</h2><br />
<br><br><br />
1. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Entry clones </Td><Td> 50-150 ng/reaction (1-7 μL) </Td></Tr><br />
<tr><td> Destination vector </td><td> 150ng/reaction (1 μL) </td></tr><br />
<Tr><Td> TE Buffer </Td><Td> to 8 − 9 μL </Td></Tr><br />
</Table><br />
<br><br />
2. Vortex LR ClonaseII enzyme mix briefly. Add 1 – 2 μL, to the components above <br />
<br><br />
and mix well by vortexing and spin down. <br />
<br><br />
3. Incubate reaction at 25˚C for 16 hours.<br />
<br><br />
4. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.<br />
<br><br />
5. Incubate at 37˚C for 10 minutes.<br />
<br><br />
<br><br />
<br />
<br />
<h2>Purification of plasmid DNA by High Pure PCR Product Kit</h2><br />
<br><br />
1. Add 500 uL of Binding buffer to DNA sample.<br />
<br><br />
2. Apply the sample to the Spin column. Centrifuge the column at 20,000 x g for 1 min.<br />
<br><br />
3. Discard the flow through fraction. Add 500 uL of Binding buffer to the column and centrifuge at 13,000 x g for 1 min.<br />
<br><br />
4. Discard the flow through fraction. Add 200 uL of Binding buffer to the column and centrifuge at 13,000 x g for 1 min.<br />
<br><br />
5. Discard the flow through fraction. Add 50 uL of Elution buffer to the column and centrifuge at 13,000 x g for 1 min. <br />
<br><br />
Eluted sample can be collected in 1.5 mL centrifuge tube. The purified plasmid DNA is recovered in this tube.<br />
<br><br><br />
<br />
<h2>Protocol for Transfection of Adherent Cells<br />
(24 Well Plates)</h2><br />
<br><br />
1. The day before transfection, inoculate 24-well plates with an appropriate number of cells in serum-containing medium such that they will be 50 to 70% confluent the following day. For most cell lines, we recommend plating<br />
<br><br />
2.5×10<sup>5</sup> cells in 0.5 ml of medium. Incubate the cells at 37℃ in 0% incubator overnight.<br />
<br><br />
2. Fifteen to sixty minutes prior to transfection, carefully aspirate the medium from the wells and add 250 µl of fresh growth medium to each well.<br />
<br><br />
3. For each well to be transfected, prepare 25 µl of serum-free medium containing 1 pj of siLentFect as a starting point. <br />
<br><br />
4. For each well to be transfected, prepare 25 µl of serum-free medium containing DNA .Use a final concentration of 10 nM as a starting point. For example, for a 24-well plate with 250 µl of growth medium per well, prepare 25 µl of serum-free medium containing 120 nM of DNA. After mixing with the diluted siLentFect from step 3 and addition to cells, the final concentration will be 10 nM. The optimal concentration of siRNA may vary from 5 to 20 nM depending on the cell line used and the gene to be targeted.<br />
<br><br />
5. Add the diluted DNA to the diluted siLentFect. Mix by tapping or pipetting. <br />
<br><br />
Incubate 20 minutes at room temperature. <br />
<br><br />
6. Add 50 µl of complexes directly to cells in serum-containing medium. Rock the plate back and forth to mix. <br />
<br><br />
Incubate the cells at 37℃ in incubator.<br />
<br><br />
7. Gene silencing can be monitored at the mRNA or protein levels from 4 to 72 hours after the transfection. If toxicity is a problem, change the medium 4 hours post transfection.<br />
<br><br><br />
<br />
<br />
<h2>Embryo microinjection protochol</h2><br />
<br><br />
1. w; pΔ2,3 (female-virgin) yw (male)<br />
<br><br />
- (prepare the flies from 3-4 days before)<br />
<br><br />
<br />
2. mating <br />
<br><br />
w; pΔ2,3 (female-virgin) X yw (male)<br />
<br><br />
- (keep at 25℃ for 3-4 hrs ; usually from 9:00 AM) <br />
<br><br />
3. prepare <br />
<br><br />
1) NaCl/Triton X-100, 10% Na-hypochloride, D.W. keep on ice.<br />
<br><br />
2) 50ml tube for embryo collection<br />
<br><br />
3) nylon mesh<br />
<br><br />
4) cover glass (3M tape, Cot No. W-18 pastes on the center)<br />
<br><br />
5) slide glass<br />
<br><br />
6) paraffin oil<br />
<br><br />
7) 1mg/ml DNA for micro injection<br />
<br><br />
8) glass needle<br />
<br><br />
9) Microinjection buffer<br />
<br><br />
5 mM KCl, 0.1 mM Sodium Phosphate, pH6.8<br />
<br><br />
DNA EtOH ppt, wash, dissolve to 1 mg/ml in the Microinjection buffe<br />
<br><br />
4. collect the embryos<br />
<br><br />
- wash 3-4 times with NaCl/Triton X-100<br />
<br><br />
- dechorinate with 5 % Na-hypochloride <br />
<br><br />
- wash 7-8 times with D.W.<br />
<br><br />
5. put the dechorinated embryos on the prepared cover glass then drop parafin oil onto the embryos. <br />
<br><br />
6. injection<br />
<br><br />
7. move the tape containing the injected embryos to new egg plate.<br />
<br><br />
caution: the tapes keep upside down from avoiding dry for embryo.<br />
<br><br />
8. after 1 day, move the hatched larva to new vials.<br />
<br><br />
9. mating <br><br />
the hatched adult flies X yw female or male<br />
<br><br />
10. take the flies having red eye then mate with yw.<br />
<br><br />
- screening <br />
<br><br />
<br />
</div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week7
Team:KIT-Kyoto/Notebook-week7
2012-09-26T04:15:18Z
<p>Kazuko: </p>
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<td width="800px" valign="top"><div id="MIGI"><br />
<h2>September 18th</h2><br />
<br><br />
<br />
By red eye screening, we identified the strain 7 and strain 10 to be the successfully transformed lines. Photographs for the transformed red (yellow) eye fly (strain 10) were taken.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/b/ba/HAEE.JPG" width="320" height="220"><br />
<br><br><br />
<br />
<h2>September 19th</h2><br />
<br><br />
<br />
By red eye screening, we identified the strain 13 and strain 14 to be the successfully transformed lines. Photographs for the transformed red (orange) eye fly (strain 13) were taken.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/a/a6/Strain13.JPG" width="320" height="220"><br />
<br><br><br />
Drosophila S2 cells (2.5 X 10<sup>5</sup> cells per well) were plated on the 24 well plate and cultured in the Schneider’s Drosophila medium containing 10% fetal bovine serum at 25 ℃.<br />
<br><br><br />
<br />
<h2>September 20th</h2><br />
<br><br />
By red eye screening, we identified the strain 23 and strain 29 to be the successfully transformed lines.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 21st</h2><br />
<br><br />
Red eye screening was continued.<br />
<br><br><br />
<br />
<br />
<h2>September 22nd</h2><br />
<br><br />
Red eye screening was continued.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<h2>September 23rd</h2><br />
<BR><br />
By red eye screening, we identified the strain 56 and strain 65 to be the successfully transformed lines. These transformed flies are kept at 25℃ and later will be further inspected for chromosome linkage of the transgene. <br />
<br><br><br />
<br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week6
Team:KIT-Kyoto/Notebook-week6
2012-09-26T04:14:59Z
<p>Kazuko: </p>
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</td><br />
<br />
<br />
<td width="800px" valign="top"><div id="MIGI"><br />
<br />
<h2>September 11th</h2><br />
<br><br />
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<h2>September 12th</h2><br />
<br><br />
The transfected cells were examined under the confocal lazer microscope (Olympus, Fv10i)<br />
<br><br />
Results: about 20 % cells were observed to be EGFP-positive, indicating that the P-element plasmid, pUAS-EGFP-TNFAIP3 is functional in Drosophila cells. We therefore decided to microinject this DNA into Drosophila embryos to establish transgenic flies.<br />
<br><br><br />
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 13th</h2><br />
<br><br />
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.<br />
<br><br><br />
<br />
<br />
<h2>September 14th</h2><br />
<br><br />
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible. The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.<br />
<br><br><br />
<br />
<br />
<h2>September 15th</h2><br />
<br><br />
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 16th</h2><br />
<BR><br />
<br />
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. In total 83 flies from microinjected embryos were mated with yw flies. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible.<br />
<br><br><br />
<br />
<br />
<h2>September 17th</h2><br />
<BR><br />
<br />
The progeny flies were inspected by dissecting microscope to look for the successfully transformed w+ red eye flies (red eye screening). However, no red eye fly was found.<br />
<br><br><br />
<br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week7">>>>>>>>>>WEEK7</a></div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week5
Team:KIT-Kyoto/Notebook-week5
2012-09-26T04:14:24Z
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</td><br />
<br />
<td width="800px" valign="top"><div id="MIGI"><br />
<br />
<h2>September 4th</h2><br />
<br><br />
The female-virgin flies (yw) and male flies (yw) were collected for mating with the microinjected males and females.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 5th</h2><br />
<br><br />
The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 6th</h2><br />
<br><br />
The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 7th</h2><br />
<br><br />
The eclosed male and virgin female flies from microinjected embryos were collected and kept at 25℃ for maturation.<br />
<br><br><br />
<br />
<br />
<h2>September 8th</h2><br />
<br><br />
<br />
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible. The eclosed male and virgin female flies from microinjected embryos were again collected and kept at 25℃ for maturation. The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 9th</h2><br />
<BR><br />
The eclosed male and virgin female flies from microinjected embryos were again collected and kept at 25℃ for maturation. In total 83 flies were eclosed from the microinjected embryos. The calculated viability for the microinjected flies was 12.0%. The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.<br />
<br><br />
<br><br />
Drosophila S2 cells (2.5 X 10<sup>5</sup> cells per well) were plated on the 24 well plate and cultured in the Schneider’s Drosophila medium containing 10% fetal bovine serum at 25 ℃.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 10th</h2><br />
<br><br />
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible.<br />
<br><br />
<br><br />
<br />
<br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week6">>>>>>>>>>WEEK6</a></div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week4
Team:KIT-Kyoto/Notebook-week4
2012-09-26T04:14:01Z
<p>Kazuko: </p>
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<h2>August 27th</h2><br />
<br><br />
<br />
The female-virgin flies (w; Δ2,3) and male flies (yw) were collected for microinjection. The collected flies were kept in the 25℃ incubator separately.<br />
<br><br />
In total 50 pairs were collected.<br />
<br><br><br />
Large scale purification of the pUAS-flag-TNFAIP3 (pTFW-TNFAIP3) for microinjection into Drosophila embryos. <br />
<br><br />
To purify pUAS-flag-TNFAIP3 DNA, E. coli carrying the plasmid was cultured in 100 mL of LB ampicillin(+) nedium at 37℃ for 16 hours.<br />
<br><br><br />
<br />
<h2>August 28th</h2><br />
<br><br />
1.Sequencing of pUAS-flag-TNFAIP3 (pTFW-TNFAIP3) and pTGW-TNFAIP3 DNA<br />
<br><br />
To determine nucleotide sequence of pUAS-flag-TNFAIP3 (pTFW-TNFAIP3) and pTGW-TNFAIP3 DNA, we send the DNAs to the company. The sequencing data confirmed that pUAS-flag-TNFAIP3 is properly constructed.<br />
<br><br><br />
2. Midi-prep purification of the pUAS-flag-TNFAIP3 DNA<br />
<br><br />
The pUAS-flag-TNFAIP3 DNA was purified from E. coli cultured on on August 22 according to protocol using Pure LinkTM HiPure Plasmid Midiprep. Concentration of the obtained DNA was measured and adjusted it to be 1mg/mL<br />
<br><br><br />
<br />
<br />
<br />
<br />
<br />
<h2>August 30th</h2><br />
<br><br />
Micro-injection of the pUAS-flag-TNFAIP3 DNA into Drosophila embryos<br />
<br><br />
<strong>Microinjection</strong><br />
<br><br />
1. In the morning, w; Δ2,3 (female-virgin) were crossed with yw (male) in the cage and kept for 3 to 4 hours at 25℃.<br />
<br><br />
2. NaCl/Triton X-100, 10% Na-hypochloride, H<sub>2</sub>O were kept on ice.<br />
<br><br />
3. The tape was placed on the cover glass (3M tape, Cot No. W-18 pastes on the center).<br />
<br><br />
4. parafin oil is prepared.<br />
<br><br />
5. 1mg/ml DNA in microinjection buffere is prepared.<br />
<br><br />
6. glass needles for microinjection were prepared.<br />
<br><br />
7. The early embryos were collected on the nylon mesh and washed 3-4 times with NaCl/Triton X-100. Then embryos were dechorinated with 5 % Na-hypochloride. Then the dechorionated embryos were washed 7-8 times with H<sub>2</sub>O.<br />
<br><br />
8. The dechorinated embryos were placed on the prepared cover glass then parafin oil was dropped onto the embryos.<br />
<br><br />
9. DNA (pUAS-flag-TNFAIP3) was microinjected into embryos.<br />
<br><br />
10. After microinjection, the injected embryos were removed with the tape on the cover glass and placed onto the new egg plate. The tapes keep upside down from avoiding dry the embryos. <br />
<br><br />
11. The plate was kept in the 25℃ incubator.<br />
<br><br />
In total 692 embryos were microinjected with pUAS-flag-TNFAIP3 DNA (1mg/ml in microinjection buffer).<br />
<br><br />
Microinjection buffer: 5 mM KCl, 0.1 mM Sodium Phosphate, pH6.8<br />
<br><br />
<br />
DNA was EtOH-precipitated, washed with 75% EtOH and dissolved to 1 mg/ml in the Microinjection buffer.<br />
<br><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d2/Injectionしてるとこ.JPG" width="500" height="300"><br />
<br><br><br />
<br />
<br />
<h2>August 31st</h2><br />
<br><br />
<br />
The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 1st</h2><br />
<br><br />
The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator. About 20 larvae were put into a fly food vial.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<br />
<h2>September 2nd</h2><br />
<br><br />
The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator. In total 144 larvae were collected. The hatching efficiency after microinjection was calculated to be 20.8%.<br />
<br><br><br />
<br />
<br />
<br />
<br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week3
Team:KIT-Kyoto/Notebook-week3
2012-09-26T04:13:37Z
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<td width="800px" valign="top"><div id="MIGI"><br />
<h2>August 20th</h2><br />
<br><br />
<br />
1. Isolation of DNA fragment from the gel<br />
Sample applied to the electrophoresis<br />
<br><br><br />
<strong>Composition</strong> <br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><Td> a product of PCR attB TNFAIP3(8/1) </Td></Tr><br />
<Tr><Td> DNA sample </Td><Td> 90μL</Td></Tr><br />
<Tr><Td> 6×Dye </Td><Td> 18μL</Td></Tr><br />
<Tr><Td> Total </Td><Td> 108μL</Td></Tr><br />
</Table><br />
<br><br />
<br />
<br><br />
<strong>Results</strong><br><br><br />
<img src="https://static.igem.org/mediawiki/2012/b/b8/0820.png" width="500" height="300"><br />
<br><br><br />
DNA was isolated from the agarose gel by QIA Quick Gel Extraction Kit, then the DNA was dissolved in 40 uL of TE.<br />
<br><br />
<br><br />
<br />
<h2>August 21st</h2><br />
<br><br />
<br />
1. Measuring the concentration of the attB TNFAIP3 DNA fragment<br />
<br><br />
The order of sample applied to the electrophoresis<br />
<br><br />
1kbmarker3uL,1uL of 5 fold dilution of attB TNFAIP3, 1uL of 10 fold dilution of attB TNFAIP3 DNA fragment,2uL of 1kbmarker, 1uL of 15 fold dilution of attB TNFAIP3 DNA fragment, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragment, 1kbmarker1uL, <br />
<br />
<br><br><br />
<strong>Result</strong><br />
<br><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/67/0821kit.png" width="500" height="300"><br />
<br><br><br />
The concentration of the isolated attB TNFAIP3 DNA fragments was estimated to be 35ng/uL.<br><br><br />
<br><br><br />
2. BP reaction<br><br />
1.5mL tube<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> attB TNFAIP3(35ng/μL)</Td><Td> 2μL </Td></Tr><br />
<Tr><Td> pDONR(150ng/μL) </Td><Td> 1μL </Td></Tr><br />
<Tr><Td> TE buffer </Td><Td> 5μL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 8μL </Td></Tr><br />
</Table><br />
<br><br><br />
We added 2uL of BP Clonase Ⅱ enzyme mix to this solution and incubate it for 2 hour. <br />
<br><br><br />
<br />
3. Transformation<br><br />
We added 100uL of XL1-Blue to the BP reaction products to do transformation.<br />
<br />
<br />
<br><br />
<br><br />
<br />
<h2>August 22nd</h2><br />
<br><br />
<br />
We isolated 6 colonies from a plate and incubated in 2.5mL of Kanamycin(+) LB liquid culture medium for 16 hours.<br />
<br />
<br />
<br><br><br />
<br />
<h2>August 23rd</h2><br />
<br><br />
1 Purification of the candidate pENTR-TNFAIP3 DNA<br />
<br><br />
We purified the candidate pENTR-TNFAIP3 DNA fro six independent colonies cultured on August 22 using QIA prep Spin Miniprep <br />
<br><br><br />
2 Characterization of the candidate pENTR-TNFAIP3 DNA<br />
<br><br />
We conducted PCR on the following condition to confirm the pENTR-TNFAIP3 DNA using primer designed for attB<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> BP TNFAIP3 </Td><Td> 0.2μL </Td></Tr><br />
<Tr><Td> 10× rTaq buffer </Td><Td> 0.8μL </Td></Tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 25mM MgCl<sub>2</sub> </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 10P 5'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> 10P 3'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> rTaq </Td><Td> 0.4μL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 13.4μL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 20μL </Td></Tr><br />
</Table><br />
<br><br><br />
Reaction<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> BP TNFAIP3 </Td><Td> 0.2μL </Td></Tr><br />
<Tr><Td> 10× rTaq buffer </Td><Td> 0.8μL </Td></Tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 25mM MgCl<sub>2</sub> </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 10P 5'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> 10P 3'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> rTaq </Td><Td> 0.4μL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 13.4μL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 20μL </Td></Tr><br />
</Table><br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> temperature </Td><Td> time </Td><Td> cycle </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 2min. </Td><Td> </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 15sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 60°C </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> </Td></Tr><br />
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td> </Td></Tr><br />
</Table><br />
<br><br />
<br><br />
The PCRproducts were applied to agarose gel electrophoresis <br />
<br><br />
From left to right: pDONR DNA 1uL, 6the candidate pENTR-TNFAIP3 2uL each<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/81/0823.png" width="500" height="300"><br />
<br><br />
<br><br />
The amplified DNA fragments were detected in the gel.<br />
<br><br><br />
3. LR reaction<br />
<br><br />
LR reactions were carried out under conditions as described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pENTR-TNFAIP3 (prepared on 8/23) </Td><Td> 1μL </Td></Tr><br />
<Tr><Td> pTFW or pTGW (Destination vectors) </Td><Td> 0.5μL </Td></Tr><br />
<Tr><Td> TE Buffer </Td><Td> 6.5μL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 8μL </Td></Tr><br />
</Table><br />
<br><br><br />
2uL LR clonaseⅡ enzyme mix was added to the reaction and incubated for 2.5 hour.<br />
<br><br><br />
4. The LR reaction products were transformed into E. coli XL1-Blue100uL according to the protocol and spread on the LB ampicillin(+) plate and cultured for 16 hours at 37℃.<br />
<br><br><br />
<br />
<h2>August 24th</h2><br />
<br><br />
Single colonies were isolated from a plate with the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 and cultured them in LB ampicillin(+) liquid medium for 16 hours at 37℃.<br />
<br><br><br />
<br />
<h2>August 25th</h2><br />
<br><br />
1. Purification of the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 DNA<br />
<br><br />
the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 were purified by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
2. Characterization of the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3<br />
<br><br />
We used the primer attB to confirm the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 for PCR reactions under conditions described below.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pTFW- or pTGW-TNFAIP3 </Td><Td> 0.2μL </Td></Tr><br />
<Tr><Td> 10× rTaq buffer </Td><Td> 0.8μL </Td></Tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 25mM MgCl<sub>2</sub> </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 10P 5'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> 10P 3'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> rTaq </Td><Td> 0.4μL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 13.4μL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 20μL </Td></Tr><br />
</Table><br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> temperature </Td><Td> time </Td><Td> cycle </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 2min. </Td><Td> </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 15sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 60°C </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> </Td></Tr><br />
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td> </Td></Tr><br />
</Table><br />
<br><br />
<br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/0826.png" width="500" height="300"><br />
<br><br><br />
Results: Since the amplified DNAs with appropriate sizes were detected on the gel, pTFW-TNFAIP3 and pTGW-TNFAIP3 were appeared to be successfully constructed by the Gateway system ! At this point we renamed the pTFW-TNFAIP3 as pUAS-flag-TNFAIP3.<br />
<br><br><br />
<br />
<h2>August 26th</h2><br />
<br><br />
<br />
The female-virgin flies (w; Δ2,3) and male flies (yw) were collected for microinjection. The collected flies were kept in the 25℃ incubator separately.<br />
<br><br><br />
<br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week4">>>>>>>>>>WEEK4</a></div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week2
Team:KIT-Kyoto/Notebook-week2
2012-09-26T04:13:11Z
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<br />
<td width="800px" valign="top"><div id="MIGI"><br />
<h2>August 8th</h2><br />
<br><br />
<br><br />
We isolated 5 colonies from the LB plate, and cultured.<br><br />
<br><br />
<br><br />
<br />
<br />
<h2>August 9th</h2><br />
<br><br />
<br><br />
<br />
1. Purification of the candidate pUAS-flag-TNFAIP3 DNA<br><br />
<br><br />
The candidate pUAS-flag-TNFAIP3 DNAs from five independent sample -1,-2,-3,-4 and -5 were purified by the alkaline-lysis method.<br><br />
We added 0.2uL of 10ng/uL RNase A to them and incubate them for 1 hour.<br><br />
<br><br />
<br><br />
2. Characterization of the candidate pUAS-flag-TNFAIP3 DNA<br><br />
<br><br />
The candidate pUAS-flag-TNFAIP3 DNA was digested wit EcoRⅠ as follows.<br><br />
<br> <br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Each diluted DNA sample </Td><Td> 1μL </Td></Tr><br />
<Tr><Td> 10×H buffer </Td><Td> 0.5μL </Td></Tr><br />
<Tr><Td> EcoRⅠ </Td><Td> 0.2μL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3.3μL </Td></Tr><br />
<Tr><Td> total </Td><Td> 5μL </Td></Tr><br />
</Table><br />
<br><br />
<br><br />
<br />
After incubation of these samples for 1hour, we added 1uL of 6×Dye to each of them and electrophoresed in 0.7% agarose gel in following order.<br> <br />
Left, 10fold dilution, 2uL marker, 20fold dilution, 30fold dilution, 1uL marker, 40fold dilution, 0.5uL marker right.<br><br />
<br><br />
<br><br />
<br />
<br />
<strong>Result</strong><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9b/0809.png" width="500" height="300"><br />
<br><br><br />
The concerntration of the candidate pUAS-flag-TNFAIP3 DNA was estimated as 200ng/uL.<br><br />
<br><br />
<br><br />
3. PCR amplification of the insert DNA<br><br />
<br><br />
The four 5'primers were designed for PCR as follows.<br><br />
<br><br />
<br />
・GW3r<br><br />
5’-ATCGAGGCCTGTCTAGAGAAGC<br><br />
・TNFA-1<br><br />
5’-GGCTTTGCTATGATACTCGGAACTG<br><br />
・TNFA-2<br><br />
5’-GTAAAATGTGAAACGCCCAACTGC<br><br />
・TNFA-3<br><br />
5’-GGACTCCAGAAAACAAGGGCTTT<br><br />
<br><br />
<br><br />
<br />
<br />
The 3’ primer was designed as follows.<br><br />
・SVr<br><br />
5’-GGCATTCCACCACTGCTCCC<br><br />
<br><br />
<br><br />
<br />
<br />
PCR reactions with the following combinations of primers were carried out.<br><br />
sample1→GW3r―SVr<br><br />
sample2→TNFA-1―SVr<br><br />
sample3→TNFA-2―SVr<br><br />
sample4→TNFA-3―SVr<br><br />
<br />
<br><br />
<br><br />
<br />
<br />
PCR was carried out in the following reactions.<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> </Td><Td> Each sample </Td></Tr><br />
<Tr><Td> 50ng/μL LR TNFA-1 </Td><Td> 1μL </Td></Tr><br />
<Tr><Td> 10×rTaq buffer </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 25mM MgCl<sub>2</sub> </Td><Td> 0.8μL </Td></Tr><br />
<Tr><Td> 10P 5'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> 10P 3'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> rTaq </Td><Td> 0.4μL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 12.6μL </Td></Tr><br />
<Tr><Td> total </Td><Td> 20μL </Td></Tr><br />
</Table><br />
<br><br><br />
<br />
Reaction conditions of PCR<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> temperature </Td><Td> time </Td><Td> cycle </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 2min </Td><Td> </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 15sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 55°C </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> </Td></Tr><br />
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td> </Td></Tr><br />
</Table><br />
<br><br />
<br />
<br />
<br />
<br />
<h2>August 10th</h2><br />
<br><br />
<br><br />
<br />
Electrophoresis of PCR products was carried out.<br><br />
After adding 2uL of 6×Dye to each 10uL of sample, we electrophoresed them in 0.7% agarose gel in following order.<br><br />
<br><br />
Left marker5uL sample1 sample2 sample3 sample4 Wright<br><br />
<br><br />
<br />
<strong>Result</strong><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/8/84/0810.png" width="500" height="300"><br />
<br><br />
The sizes of the PCR products were unexpected ones. We therefore concluded the construction of pUAS-flag-TNFAIP3 DNA was not successful.<br><br />
<br><br />
<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<h2>August 13th</h2><br />
<br><br />
<br><br />
<br />
Cut check of the plasmid DNA(the candidate pENTR-TNFAIP3) after BP reaction.<br><br />
<br><br />
The candidate pENTR-TNFAIP3 was digested with HindⅢ that will cut out the insert DNA.<br><br />
<br><br />
<br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> the candidate pENTR-TNFAIP3 prepared on 8/6 </Td><Td> 1μL </Td></Tr><br />
<Tr><Td> 10×M buffer </Td><Td> 0.5μL </Td></Tr><br />
<Tr><Td> Hind Ⅲ </Td><Td> 0.2μL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3.3μL </Td></Tr><br />
<Tr><Td> total </Td><Td> 5μL </Td></Tr><br />
</Table><br />
<br><br />
<br>After reaction, samples were electrophoresed in following order.<br><br />
<br><br />
Left: 1kb marker5uL cut sample <br><br />
Right: uncut sample<br><br />
<br><br />
<br><br />
<strong>Results</strong><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/2a/0813.png" width="500" height="300"><br />
<br><br><br />
Insert size was different from the expected one. We therefore concluded that the construction of entry DNA was not successful.<br><br />
<br />
<br><br />
<br><br />
<br />
<br />
<h2>August 14th</h2><br />
<br><br />
<br><br />
<br />
Based on the results, we decided to carry out the PCR reaction for TNFAIP3 DNA again.<br><br />
<br><br />
<br />
Composition of the reaction <br />
<Table Border Cellspacing="0"><br />
<Tr><Td> </Td><Td> sample1 </Td></Tr><br />
<Tr><Td> 10ng/μL TNFAIP3 </Td><Td> 6μL </Td></Tr><br />
<Tr><Td> 10×KOD plus buffer </Td><Td> 10μL </Td></Tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 10μL </Td></Tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 3.2μL </Td></Tr><br />
<Tr><Td> 10P 5'primer </Td><Td> 3μL </Td></Tr><br />
<Tr><Td> 10P 3'primer </Td><Td> 3μL </Td></Tr><br />
<Tr><Td> KOD plus </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 62.8μL </Td></Tr><br />
<Tr><Td> total </Td><Td> 100μL </Td></Tr><br />
</Table><br />
<br><br />
<br><br />
<br />
Reaction conditions<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> temperature </Td><Td> time </Td><Td> cycle </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 2min </Td><Td> </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 15sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 60°C </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> </Td></Tr><br />
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td> </Td></Tr><br />
</Table><br />
<br><br />
<br><br />
PCR product was applied to the 0.7% agarose gel electrophoresis.<br><br />
<br><br />
<br />
<strong>Results</strong><br><br />
<img src="https://static.igem.org/mediawiki/2012/c/c4/0814.png" width="500" height="300"><br />
<br><br />
<br />
<br><br />
<br><br />
<br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week3">>>>>>>>>>WEEK3</a></div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week1
Team:KIT-Kyoto/Notebook-week1
2012-09-26T04:12:52Z
<p>Kazuko: </p>
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<td width="800px" valign="top"><div id="MIGI"><br />
<h2>August 1st</h2><br />
<br><br />
<br><br />
<br />
Since it takes at least one month to establish transgenic flies, we decided to first construct the P-element plasmid for microinjection into Drosophia embryos.<br><br />
<br><br />
We performed PCR in the following conditions.<br><br />
<br><br />
<br />
TNFAIP3<br><br />
Composition for reaction<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> </Td><Td> sample1 </Td></Tr><br />
<Tr><Td> 10ng/μL TNFAIP3 </Td><Td> 6μL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 10μL </Td></Tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 10μL </Td></Tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 3.2μL </Td></Tr><br />
<Tr><Td> 10P 5'primer </Td><Td> 3μL </Td></Tr><br />
<Tr><Td> 10P 3'primer </Td><Td> 3μL </Td></Tr><br />
<Tr><Td> KOD plus </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 62.8μL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 100μL </Td></Tr><br />
</Table><br />
<br><br />
<br />
Reaction conditions<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> temperature </Td><Td> time </Td><Td> cycle </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 2min. </Td><Td> </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 15sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 60°C </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> </Td></Tr><br />
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td> </Td></Tr><br />
</Table><br />
<br><br />
<br><br />
<br />
<br />
<h2>August 2nd</h2><br />
<br><br />
<br><br />
<br />
1. PCR product check<br><br />
<br><br />
We run 0.7% agarose gel electorophoresis in the following conditions.<br />
<br><br><br />
<strong>Composition</strong><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> </Td><Td> 8/1 the attB TNFAIP3 PCR products </Td></Tr><br />
<Tr><Td> DNA sample </Td><Td> 10μL </Td></Tr><br />
<Tr><Td> 6×Dye </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> total </Td><Td> 12μL </Td></Tr><br />
</Table><br><br />
<br><br />
<br />
The order of sample application<br />
<br><br />
Left to right: 1kb marker(5uL), attB TNFAIP3, attB API2-MALT1-1, -2, -3, -4<br />
<br><br><br />
<br />
<strong>Results</strong><br />
<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/e0/0802.png" width="500" height="300"><br />
<br><br />
<br><br />
<br />
2. DNA purification from gel<br><br />
We electrophoresed in 0.7% agarose gel in the following conditions.<br><br />
<br><br />
<br />
<strong>Composition</strong><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> </Td><Td> 8/1 attB TNFAIP3 PCRproduct </Td></Tr><br />
<Tr><Td> DNA sample </Td><Td> 90μL </Td></Tr><br />
<Tr><Td> 6×Dye </Td><Td> 18μL </Td></Tr><br />
<Tr><Td> total </Td><Td> 108μL </Td></Tr><br />
</Table><br />
<br><br><br />
We applied half of 108 uL of sample to each of two lanes of Agaroe gel with 5uL of 1kb marker.Then we detected the DNA band in the gel.<br><br><br />
<br />
<strong>Results</strong><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/19/0802-2.png" width="500" height="300"><br />
<br><br />
<br />
We purifed DNA from the gel by QIA Quick Gel Extraction Kit.<br><br />
<br><br><br />
・Eatimation of the amount of attB TNFAIP3 DNA fragments by electrophoresis.<br><br />
Order of samples applied to the agarose gel<br><br />
The samples were electrophoresed in 0.7% agarose gel in following order.<br><br><br />
Right to left:3uL of 1kb marker 1uL of 5 fold dilution of attB TNFAIP3 DNA fragments,1uL of 10 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 1kb marker.<br><br />
<br><br />
<br />
<strong>Results</strong><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/0/0e/0802-3.png" width="500" height="300"><br />
<br><br />
We have estimated amount of the attB TNFAIP3 DNA to be 80ng/uL.<br><br />
<br><br />
<br><br />
<br />
<br />
<h2>August 3rd</h2><br />
<br><br />
<br><br />
1. BP reaction<br><br />
<br><br />
<br />
BP reaction was carried out in the following conditions.<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> attB TNFAIP3(80ng/μL) </Td><Td> 1μL </Td></Tr><br />
<Tr><Td> pDONR(455ng/μL) </Td><Td> 0.35μL </Td></Tr><br />
<Tr><Td> TE buffer </Td><Td> 7.65 </Td></Tr><br />
<Tr><Td> total </Td><Td> 9μL </Td></Tr><br />
</Table><br />
<br><br />
<br><br />
One uL of BP Clonase Ⅱ enzyme mix was added to this solution and incubated for 2.5 hours.<br><br />
<br><br />
<br><br />
2. Transformation of E.coli by BP reaction products<br><br />
2uL of BP TNFA reaction product was added to 100uL of E.coli XL1-Blue to do transform ation.<br><br />
At last, cells were divided into 10,40,100,and 200 uL,and spreaded on LB Kanamycin(+)plate,Plats wera incubated for 16 hours at 37°C.<br><br />
<br><br />
<br><br />
<br />
<br />
<h2>August 4th</h2><br />
<br><br><br />
<br />
Four independent 4 colonies were picked up from the plate incubated from 8/3 as described in ②,then cultured in 2.5mL of LB Kanamycin(+) liquid culture medium for 16 hours.<br><br />
<br><br />
<br><br />
<br />
<br />
<h2>August 5th</h2><br />
<br><br />
<br><br />
<br />
Plasmid DNAs were isolated from E.coli cultured in 4 of LB liquid culture medium which we made on 8/4 by the alkaline-lysis method, we added the DNA to 20uL TE and added 10uL of the solution to 2uL of 6×Dye. Then we electrophoresed it with marker, 2uL of pDONR+ 2uL of 6×Dye+ 8uL of TE,in 0.7% agarose gel in following order (from left to right)<br><br />
<br><br />
Left control pDONR DNA, BP TNFA-1,-2,-3,-4<br><br />
<br><br />
<br />
<strong>Results</strong><br><br />
<img src="https://static.igem.org/mediawiki/2012/3/31/0805.png" width="500" height="300"><br><br />
<br><br />
<br><br />
<br />
<h2>August 6th</h2><br />
<br><br />
<br><br />
E.coli carrying the plasmid sample -1 and -2 in LB Kanamycin(+)liquid culture medium for 16 hours.<br><br />
<br><br />
<br><br />
<br />
<h2>August 7th</h2><br />
<br><br />
<br><br />
1. Purification of candidate pENTR-TNFAIP3<br><br />
We purified candidate plasmid-1 and -2 by QIA Prep Spin Miniprep Kit, dissolved it in 30uL of Buffer EB.<br><br />
Then,the samples were electrophoresed in 0.7% agarose gel.<br><br />
<br><br />
<br />
We made samples as follows.<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> </Td><Td> pDONR </Td><Td> BP TNFA-1 </Td><Td> BP TNFA-2 </Td></Tr><br />
<Tr><Td> DNA sample </Td><Td> 1μL </Td><Td> 1μL </Td><Td> 1μL </Td></Tr><br />
<Tr><Td> 6×Dye </Td><Td> 1μL </Td><Td> 1μL </Td><Td> 1μL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 4μL </Td><Td> 4μL </Td><Td> 4μL </Td></Tr><br />
<Tr><Td> total </Td><Td> 6μL </Td><Td> 6μL </Td><Td> 6μL </Td></Tr><br />
</Table><br />
<br><br />
<br><br />
We electrophoresed them in following order.<br><br />
Left marker 5uL control pDONR DNA ,pENTR-TNFAIP3-1 DNA,pENTR-TNFAIP3 -2 DNA marker 4uL marker 3uL Wright.<br><br />
<br><br />
<strong>Results</strong><br><br />
<img src="https://static.igem.org/mediawiki/2012/2/2e/0807.png" width="500" height="300"><br />
<br><br />
Concentration of pENTR-TNFAIP3-2 DNA was estimated at around 100ng/uL.<br><br />
<br><br />
<br><br />
2. LR reaction<br><br />
We made following solution(vials)in 1.5mL tube.<br><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> BP TNFA-2 </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> pTFW(Destination vector) </Td><Td> 0.5μL </Td></Tr><br />
<Tr><Td> TE buffer </Td><Td> 6.5μL </Td></Tr><br />
<Tr><Td> total </Td><Td> 9μL </Td></Tr><br />
</Table><br />
<br><br />
we added 1uL of LR clonaseⅡ enzyme mix to this solution and incubate it for 2.5 hours.<br><br />
<br><br />
3. Transformation of E.coli with LR reaction products.<br><br />
Transformation of E.coli was carried out by adding 2uL of LR reaction products to 100uL of XL1-Blue and plated on the LB ampicillin(+)plate and cultured at 37°C.<br><br />
<br><br />
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http://2012.igem.org/Team:KIT-Kyoto/Notebook-week5p
Team:KIT-Kyoto/Notebook-week5p
2012-09-26T04:12:29Z
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<h2>September 20th</h2><br />
<br><br />
At 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP DNA was added to the well. As a negative control, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng of pUAS-EGFP DNA alone was added to the well. Similarly, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-LacZ DNA was added to the well. As a negative control for this experiment, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng of pUAS-LacZ DNA alone was added to the other well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 or 72 hours at 25 ℃.<br />
<br><br><br />
<br />
<h2>September 22nd</h2><br />
<br><br />
The cells transfected with pAct5C-GAL4 DNA and pUAS-EGFP DNA were examined under the confocal lazer microscope (Olympus, Fv10i).<br />
<br><br />
Results: 12.6% cells were observed to be EGFP-positive, while only o.5% cells were apparently EGFP-positive for the negative control cells, indicating that the P-element plasmid, pUAS-EGFP DNA, Biobrick part (BBa_K758006) is truly functional in Drosophila cells. We therefore decided to send this prasmid to iGEM Headquarters.<br />
<br><br><br />
Leftpanel: cells co-transfected with pAct5C-GAL4 DNA and pUAS-EGFP DNA<br />
<br><br />
Right panel: cells transfected with pUAS-EGFP DNA alone (negative control)<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/b/b5/実験ノート1.png" width="282" height="282"> <img src="https://static.igem.org/mediawiki/2012/c/cd/実験ノート2.png" width="282" height="282"><br />
<br><br><br />
The cells transfected with pAct5C-GAL4 DNA and pUAS-LacZ DNA were fixed with cold methanol for 20 min or 4% paraformaldehyde for 15 min. After fixation, cells were washed with 0.5% Triton X-100 in PBS, then masked with 5% normal goat serum in PBS for 1 hour. Then the monoclonal anti-beta galactosidase antibody (X500 dilution in PBS containing 5% normal goat serum) was added to the fixed cells and incubated for 16 hours at 4℃. <br />
<br><br><br />
<br />
<br />
<br />
<h2>September 23rd</h2><br />
<BR><br />
<br />
By red eye screening, we identified the strain 56 and strain 65 to be the successfully transformed lines. These transformed flies are kept at 25℃ and later will be further inspected for chromosome linkage of the transgene. <br />
<br><br><br />
After incubation with the first antibody, the cells were washed with PBS (three times for 10 min each). Then the Alexa-conjugated anti-mouse IgG (X400 dilution in PBS containing 5% normal goat serum) added to the cells and incubate for 4 hours at 4℃. After washing with PBS (three times for 10 min each), cells were mounted with mounting medium in DAPI and examined under the confocal lazer microscope (Olympus, Fv10i).<br />
Results: 10.1% cells co-transfected with pAct5C-GAL4 DNA and pUAS-LacZ DNA were observed to be LacZ-positive, while no LacZ-positive cell was detected for the negative control cells, indicating that the P-element plasmid, pUAS-LacZ DNA, Biobrick part (BBa_K758005) is truly functional in Drosophila cells. We therefore decided to send this plasmid to iGEM Headquarters. <br />
<br><br><br />
After 72 hours incubation, the cells transfected with pAct5C-GAL4 DNA and pUAS-EGFP DNA were examined under the confocal lazer microscope (Olympus, Fv10i). Results were nearly the same as those after 48 hours incubation.<br />
<br><br><br />
Leftpanel: cells co-transfected with pAct5C-GAL4 DNA and pUAS-LacZ DNA<br />
<br><br />
Right panel: cells transfected with pUAS-LacZ DNA (negative control)<br />
<br><br><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week4p
Team:KIT-Kyoto/Notebook-week4p
2012-09-26T04:11:59Z
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<td width="800px" height="510px" valign="top"><br />
<div id="MIGI"><br />
<h2>September 13th</h2><br />
<br><br />
<strong>1-1-50 and 2-2-8 Isolating a single colony of E. coli carrying the candidate pSV1C3-UAS-LacZ or pSB1C3-HS-GAL4</strong><br />
<br><br />
We isolated colonies (one for pSB1C3-UAS-LacZ ,three for pSB1C3-HS-G4L4) and cultured in liquid medium(2.5ml LB Chloramphenicol(+)) at 37℃ for 16 hours. No transformed colony was detected for E. coli carrying the candidate pSB1C3-Act5C-GAL4.<br />
<br><br><br />
<strong>1-2-8 Cut check for the candidate pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP</strong><br />
<br><br />
The candidate pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP DNAs (made 9/12) were digested with EcoRⅠ/ SpeⅠ and BglⅡ, respectively.<br />
<br><br><br />
EcoRⅠand SpeⅠ<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA sample </Td><Td> 1uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 0.5uL </Td></tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.2uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.2uL </Td></Tr><Tr><br />
<Td> 100×BSA </Td><Td> 0.05uL </Td></Tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 3.25uL </Td></tr><tr><br />
<td> Total </td><Td> 5uL </Td></tr><br />
</Table><br />
<BR><BR><br />
BglⅡ<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA sample </Td><Td> 1uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><Td> 0.5uL </Td></tr><br />
<Tr><Td> BglⅡ </Td><Td> 0.2uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><Td> 3.3uL </Td></tr><br />
<td> Total </td><Td> 5uL </Td></tr><br />
</Table><br />
<br><br><br />
Then the digested samples were applied to Agarose gel electrophoresis in the order of no cut sample, EcoRⅠ/SpeⅠ-digested sample and BglⅡ-digested sample.<br />
<br><br><br />
Result of electrophoresis<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/3/3f/0913kit.png" width="500" height="300"><br />
<br><br><br />
Results: No insert DNA was detected. Therefore we were not successful for these cloning.<br />
<br><br><br />
<strong>1-1-51, 2-1-37 and 2-2-9. Ligation</strong><br />
<br><br />
Ligation of DNA fragments carrying GAL4 to pSB1C3 DNA was carried out for 2 hours at 16℃ in the reaction described below.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (double digested with BglⅡand SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (double digested with BglⅡand SpeⅠ) </td><Td> 1.5uL </Td></tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 2.5uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 2.5uL </Td></tr><tr><br />
<td> Total </td><Td> 7.5uL </Td></tr><br />
</Table><br />
<br><BR><br />
Ligation of DNA fragments carrying G4L4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to SB1C3 DNA was carried out for 1 hours at 16℃ in the reaction described below.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3(PCR)(double digested with XbaⅠand SpeⅠ) </Td><Td> 2.5uL </Td></Tr><br />
<tr><td> GAL4(double digested with BglⅡand SpeⅠ) </td><Td> 2.5uL </Td></tr><br />
<tr><td> HS or Act5c fragments (double digested with XbaⅠand BamHⅠ) </td><Td> 2uL </Td></tr><br />
<td> Ligation high </td><Td> 7uL </Td></tr><tr><br />
<td> Total </td><Td> 14uL </Td></tr><br />
</Table><br />
<br><BR><br />
Ligation of DNA fragments carrying EGFP or LacZ to pSB1C3-UAS was carried out for 1 hours at 16℃ in the reaction described below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS (double digested with BglⅡand SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> EGFP fragments or LacZ fragments (double digested with BglⅡand SpeⅠ) </td><Td> 2uL </Td></tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 2uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 5uL </Td></tr><tr><br />
<td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-52, 2-1-38 and 2-2-10 Transformation of E. coli by Ligation products</strong><br />
<br><br />
We did transformation of E. coli Xl-1 blue with each of ligation products. Finally, we spread them on the LB Chloramphenicol(+) plate and cultured at 37℃ for 16 hours.<br />
<br><br><br />
<br />
<h2>September 14th</h2><br />
<br><br />
<strong>1-1-53 and 2-1-39 Isolating a single colony of E. coli transformed with Ligation products</strong><br />
<br><br />
We picked up three colonies of E. coli carrying the candidate pSB1C3-G4L4, two from the candidate pSB1C3-UAS-EGFP and three from the candidate pSB1C3-UAS-LacZ (made on 9/13), and cultured in 2.5mL LB Chloramphenicol(+) liquid medium at 37℃ for 16 hours.<br />
<br><br><br />
<strong>1-1-54 and 2-2-11 Purification of the candidate pSB1C3-UAS-LacZ and pSB1C3-HS-GAL4 DNA</strong><br />
<br><br />
The pSB1C3-UAS-LacZ DNA and pSB1C3-HS-G4L4 DNA was purified from E. coli (cultivated on 9/12) by QIA prep Spin Miniprep Kit. <br />
<br><br><br />
<strong>1-1-55 and 2-2-12 Cut check of the candidate pSB1C3-UAS-LacZ, pSB1C3-UAS-EGFP, and pSB1C3-HS-GAL4 DNA</strong><br />
<br><br />
These candidate pSB1C3-UAS-EGFP DNA (prepared on 9/12), pSB1C3-UAS-LacZ DNA (prepared on 9/12), pSB1C3-UAS-LacZ DNA (prepared on 9/14) and pSB1C3-HS-G4L4 DNA (prepared on 9/14)<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA sample </Td><Td> 1uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 1uL </Td></tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.2uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.2uL </Td></Tr><Tr><br />
<Td> 100×BSA </Td><Td> 0.1uL </Td></Tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 7.5uL </Td></tr><tr><br />
<td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><br><br />
Digested samples were applied to the Agarose gel electrophoresis.<br />
<br><br />
From the left side,<br><br />
Two pSB1C3-UAS-EGFP DNA isolated from independent colonies (9/12) uncut , digested two pSB1C3-UAS-EGFP DNA (9/12),<br />
<br><br />
pSB1C3-UAS-LacZ DNA (9/12) uncut, digested pSB1C3-UAS-LacZ DNA (9/12)cut,<br />
<br><br />
pSB1C3-UAS-LacZ DNA (9/14) uncut, digested pSB1C3-UAS-LacZ DNA (9/14), <br />
<BR><br />
pSB1C3-HS-G4L4 DNA from three independent colonies (9/14) uncut, digested pSB1C3-HS-G4L4 DNA from three independent colonies (9/14)<br />
<br><br><br />
<br />
Results<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/ec/0914kit.png" width="500" height="300"><br />
<br><br><br />
We found that pSB1C3-UAS-LacZ DNA (9/14) was successfully constructed !. This is the first one we successfully constructed as a Biobrick part.<br />
<br><br><br />
<strong>2-2-13 Ligation</strong><br />
<br><br />
Ligation of DNA fragments carrying G4L4 and those carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA was carried out for 2 hours at 16℃ in a reaction described below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3(PCR) (double digested with XbaⅠand SpeⅠ) </Td><Td> 2uL </Td></Tr><br />
<tr><td> GAL4 (double digested with BglⅡand SpeⅠ) </td><Td> 3uL </Td></tr><br />
<tr><td> HS fragments or Act5c fragments (double digested with XbaⅠand BamHⅠ) </td><Td> 1uL(HS) or 2.5uL(Act5c) </Td></tr><tr><br />
<td> Ligation high </td><Td> 6uL(HS) or 7.5uL(Act5c) </Td></tr><tr><br />
<td> Total </td><Td> 12uL(HS) or 15uL(Act5c) </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>2-2-14. Transformation of Ligation products</strong><br />
<br><br />
Ligation products were transformed into E. coli XL-1 blue.<br />
<br />
<br><br><br />
<br />
<h2>September 15th</h2><br />
<br><br />
<strong>1-1-56. Purification of candidate plasmid DNAs</strong><br />
<br><br />
We reproduced The candidate pSB1C3-UAS-LacZ DNA was purified from the three independent colonies, pSB1C3-UAS-EGFP DNA from two independent colonies, pSB1C3-G4L4 DNA from three independent colonies by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
Cut check of the candidate pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA<br />
<br><br />
The candidate pSB1C3-UAS-LacZ DNA from two independent colonies, the candidate pSB1C3-UAS-EGFP DNA from two independent colonies and pSB1C3-G4L4 DNA from three independent colonies were double digested with EcoRI and SpeI. <br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA sample </Td><Td> 1uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 1uL </Td></tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.2uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.2uL </Td></Tr><Tr><br />
<Td> 100×BSA </Td><Td> 0.1uL </Td></Tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 7.5uL </Td></tr><tr><br />
<td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><br><br />
The digested samples were applied to Agarose gel electrophoresis. From the left side lane to the right, two digested candidate pSB1C3-UAS-LacZ DNA, two digested candidate pSB1C3-UAS-EGFP DNA, three digested candidate pSB1C3-G4L4 are shown.<br />
<br><br><br />
Results<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d3/0915kit.png" width="500" height="300"><br />
<br><br><br />
We identified the properly constructed pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA on the gel ! These are Biobrick parts we successfully constructed.<br />
<br><br><br />
<br />
<br />
<h2>September 17th</h2><br />
<BR><br />
<strong>1-1-59 Cut check of the Biobrick part DNAs</strong><br />
<br><br />
The pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA were purified by QIA prep Spin Miniprep Kit. These DNAs were double digested with EcoRI and SpeI again and the digested samples were applied to Agarose gel electrophoresis.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/2/26/0917kit.png" width="500" height="300"><br />
<br><br><br />
Results: The correct size of inserts were detected in the double digested samples further confirming that the pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA are properly constructed.<br />
<br><br><br />
<strong>1-1-60 Submission of the Biobrick parts</strong><br />
<br><br />
The plasmids pSB1C3-UAS-LacZ, pSB1C3-UAS-EGFP, pSB1C3-G4L4 DNA and the previously prepared pSB1C3-UAS were sent out to the iGEM Headquarter via FedEx.<br />
<br><br><br />
<strong>1-1-61 Preparation of DNA for transfection into cultured Drosophila cells</strong><br />
<br><br />
E. coli carrying pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP were grown for 16 hours at 37℃.<br />
<br><br><br />
<br />
<h2>September 18th</h2><br />
<br><br />
pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP DNAs were purified by the QIA prep Spin Miniprep Kit.<br />
<br><br><br />
<br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week3p
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2012-09-26T04:10:34Z
<p>Kazuko: </p>
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<br><br />
</td><br />
<br />
<br />
<td width="800px" height="510px" valign="top"><br />
<div id="MIGI"><br />
<h2>September 7th</h2><br />
<br><br />
1. PCR amplification of UAS, UAS-TNFAIP3, pSB1C3, GAL4 DNA<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.25uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 34.15uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/0907akit.png" width="500" height="300"><br />
<br><br><br />
2. PCR products were applied to the agarose gel electrophoresis<br />
<br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 as shown below<br />
<br><br />
Results: no clearly amplified bands were detected.<br />
<br><br><br />
3. Re-estimation of the concentration of pSB1C3 and GAL4 DNA used for ligation on 9/5, 6<br />
<br><br><br />
4. SB1C3 and GAL4 DNA were applied to the agarose gel electrophoresis<br />
<br><br />
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker - 2uL, DNA 0.1uL, 1kbmarker 1uL<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/68/0907bkit.png" width="500" height="300"><br />
<br><br><br />
From these results, concentration of the pSB1C3 DNA was estimated to be 10ng/uL.<br />
<br><br />
<br><br />
GAL4<br />
<br><br />
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker 2uL, DNA 0.1uL, 1kbmarker 1uL<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/0907ckit.png" width="500" height="300"><br />
<br><br><br />
However no GAL DNA was detected. We lost the sample somewhere by some mistakes.<br />
<br><br><br />
<br />
<br />
<h2>September 8th</h2><br />
<br><br />
1. PCR amplification of UAS, UAS-TNFAIP3, pSB1C3 and GAL4 DNA<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td></td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
5. PCRproducts applied to the agarose gel electrophoresis<br />
<br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 10 uL each<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/be/0908kit.png" width="500" height="300"><br />
<br><br><br />
Only the PCR products for pSB1C3 was detectable.<br />
<br><br />
We repeated PCR for UAS and GAL4DNA by changing the annealing temperature<br />
<br><br />
(-1 indicates 55℃、-2 indicates 58℃)<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 55℃(-1) or 58℃(-2) </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<h2>September 9th</h2><br />
<BR><br />
1. PCRproducts were electrophoreses.<br />
<br><br />
Left to right: UAS-1, UAS-2, GAL4-1, GAL4-2 10 uL each<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/1/10/0909akit.png" width="500" height="300"><br />
<br><br><br />
We finally succeeded the amplification of the UAS fragments.<br />
<br><br><br />
2. Purification of pSB1C3 and UAS-1-PCR products<br />
<br><br />
PCR products were purified by High Pure PCR Product Kit<br />
<br><br><br />
3. The purified UAS fragments were digested with EcoRⅠ and SpeⅠ in the following reaction<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> UAS </Td><Td> 40uL </Td></Tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<Tr><td> SpeⅠ(NEB </td><td> 0.5uL </td></tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 3.5uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
4. The digested UAS DNA was applied to the agarose gel electrophoresis and purified from the gel by QIA quick Gel Extraction Kit<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/91/0909bkit.png" width="500" height="300"><br />
<br><br><br />
5. PCR amplification of GAL4<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<Tr><td> 2mM dNTPs <td> 5uL </td></tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><br />
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min10sec </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 2min10sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
6. PCR products were applied to the agarose gel electrophoresis as shown below.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/bb/0909ckit.png" width="500" height="300"><br />
<br><br><br />
7. pSB1C3 and UAS fragments were ligated in the following reactions.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (EcoRⅠ and SpeⅠ digested) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> UAS (EcoRⅠ and SpeⅠ digested) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3uL </Td></Tr><br />
<Tr><td> Ligation high </td><td> 2.5uL </td></tr><br />
<Tr><Td> Total </Td><Td> 7.5uL </Td></Tr><br />
</Table><br />
<br><BR><br />
The following reaction were carried out as a negative control. <br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (EcoRⅠ and SpeⅠ digested)</Td><Td> 1uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 4uL </Td></Tr><br />
<Tr><td> Ligation high </td><td> 2.5uL </td></tr><br />
<Tr><Td> Total </Td><Td> 7.5uL </Td></Tr><br />
</Table><br />
<br><BR><br />
8. 7 ligation products were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(+) plate and incubated at 37℃ for 16 hours.<br />
<br><br><br />
<br />
<h2>September 10th</h2><br />
<br><br />
At 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP-TNFAIP3 DNA was added to the well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 hours at 25 ℃.<br />
<br><br />
<br><br />
1. Single colony isolation of ligationproducts<br />
<br><br />
Ligation product prepared on 9/9 (UAS and pSB1C3) gave us some colonies and negative control sample gave us no colony. We therefore picked up four independent colonies and cultured them in 2.5mL LB Chloramphenicol (+) medium at 37℃ for 16 hours.<br />
<br><br><br />
2. Digestion of HS promoter fragment and Act5C promoter with XbaⅠ and BamHⅠ<br />
<br><br />
DNA fragments carrying HS promoter (prepared on 9/5) and those carrying Act5C promoter (prepared on 9/3) were digested with XbaⅠ and BamHⅠ.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> HS or Act5c </Td><Td> 40uL </Td></Tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<Tr><td> XbaⅠ-HF(NEB) <td> 0.5uL </td></tr><br />
<Tr><Td> BamHⅠ(NEB )</Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3.5uL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 50uL </Td></Tr><br />
</Table><br />
<br><BR><br />
Digested fragments were purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/27/0910akit.png" width="500" height="300"><br />
<br><br><br />
3. PCR amplification of GAL4 fragments<br />
<br><br />
We tried PCR under four different conditions described below. <br />
<Br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><Td> G-1 and G-2 </Td><Td> G-3 and G-4 </Td></Tr><br />
<tr><td> DNA template </td><td> 1uL </td><Td> 4uL </Td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 5uL </td><Td> 5uL </Td></tr><tr><br />
<td> 25mM MgSO<sub>4</sub> </td><td> 1.6uL </td><Td> 1.6uL </Td></tr><br />
<tr><td> 10P 5’ primer </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> 10P 3’ primer </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> KOD plus </td><td> 1uL </td><Td> 1uL </Td></tr><br />
<tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td><Td> 31.4uL </Td></tr><br />
<tr><td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 57℃(G-1 and G-3) or 59℃(G-2 and G-4) </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min10sec </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 2min10sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
4. PCR products were applied to agarose gel electrophoresis <br />
<br><br />
From left to right: 10uL each G-1, G-2, G-3, G-4<br />
<br><br />
<br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/eb/0910bkit.png" width="500" height="300"><br />
<br><br><br />
We found that GAL4 sequence was properly amplified under G-1 and G-2conditions.<br />
<brr><br />
5. PCR products were purified by High Pure PCR Product Kit.<br />
<br><br />
6. Digestion of pSB1C3 DNA with EcoRⅠ and SpeⅠ<br />
<br><br />
PCR-amplified pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ for 2 hours at 37℃.<br />
<br><br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3(PCR) </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> SpeⅠ(NEB) </td><Td> 0.5uL </Td></tr><tr><br />
<td> 2100×BSA </td><Td> 0.5uL </Td></tr><br />
<tr><td> dH<sub>2</sub>O <Td> 3.5uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
7. Digestion of EcoRI-digested UAS fragment by BglⅡ<br />
<br><br />
BglⅡ-digestion was carried out for 2 hours at 37℃ under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> UAS(EcoRⅠ-digested) </Td><Td> 13uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><Td> 2uL </Td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O <Td> 2.5uL </Td></tr><br />
<tr><td> Total </td><Td> 20uL </Td></tr><br />
</Table><br />
<br><BR><br />
8. Purification of restriction enzyme-digested pSB1C3 DNA and UAS fragment<br />
<br><br />
restriction enzyme-digested pSB1C3 DNA and UAS fragment (prepared on 9/10) were purified by QIA quick Gel Extraction Kit. <br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/86/0910ckit.png" width="500" height="300"><br />
<br><br><br />
9. PCR-amplified GAL4 fragments were digested with XbaⅠ and SpeⅠ under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 20uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 4uL </Td></tr><br />
<Tr><Td> XbaⅠ(NEB) </Td><Td> 0.7uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.7uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.4uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 14.6uL </Td></tr><br />
<tr><td> Total </td><Td> 40uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA was applied to agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/3/39/0910dkit.png" width="500" height="300"><br />
<br><br><br />
Results: XbaⅠ-digestion of GAL4 fragment gave two DNA fragments, indicating that GAL4 sequence contains XbaⅠ site.<br />
<br><br><br />
10. Digestion of GAL4 sequence with BglⅡ and SpeⅠ<br />
<br><br />
Digestion was carried out st 37℃ for 1hour under the conditions described below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 4.5uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA was purified by High Pure PCR Product Kit was further digested with Spe I at 37℃ for 1hour.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4(Bgl Ⅱ cut) </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> dH<sub>2</sub>O </td><Td> 4uL </Td></tr><br />
<tr><td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested fragments were purified by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/0910ekit.png" width="500" height="300"><br />
<br><br><br />
11. Insertion of GAL4 fragments and HS promoter or Act5C promoter-enhancer and UASfragment and EGFP sequence or LacZ sequence into the pSB1C3DNA<br />
<br><br><br />
Ligation reactions were carried out at 16℃ for 1hour under conditions described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCRproducts)(double digested with EcoRⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> UAS (double digested with EcoRⅠ and BglⅡ) </td><Td> 2uL </Td></tr><br />
<Tr><Td> EGFP or LacZ </Td><Td> 2uL </Td></Tr><br />
<tr><td> Ligation high </td><Td> 5uL </Td></tr><br />
<tr><td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (vector DNA) (double digested with XbaⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (double digested with BglⅡ and SpeⅠ) </td><Td> 2uL </Td></tr><br />
<Tr><Td>HS or Act5c (double digested with XbaⅠ and BamHⅠ)</Td><Td> 2uL </Td></Tr><br />
<tr><td> Ligation high </td><Td> 5uL </Td></tr><br />
<tr><td> Total </td><Td> 10uL </Td></tr><br />
</Table><br />
<br><BR><br />
12. Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(+) plate and cultured at 37℃for 16 hours.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 11th</h2><br />
<br><br />
1. Single colony isolation <br />
<br><br />
Single colonies were isolated from the plate prepared on 9/11 and cultured in 2.5mL LB Chloramphenicol(+) medium at 37℃ for 16 hours.<br />
<br><br><br />
2. Isolation of the candidate pSB1C3-UAS DNA<br />
<br><br />
The candidate pSB1C3-UAS DNA was purified by QIA prep Spin Miniprep Kit.<br />
<br><Br><br />
3. The purified candudate pSB1C3-UAS was digested with BglII for 2 hours.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS-1, -2, -3, -4 </Td><Td> 5uL </Td></Tr><br />
<tr><td> 3buffer(NEB) </td><Td> 2uL </Td></tr><br />
<Tr><Td> BglⅡ </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><Td> 12.5uL </Td></tr><br />
<td> Total </td><Td> 20uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were applied to agarose gel electrophoresis as shown below. Left to right: pSB1C3-1 uncut, pSB1C3 cut, -2 uncut, -2 cut, -3 uncut, -3 cut, -4 uncut, -4 cut<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/0/0c/0911akit.png" width="500" height="300"><br />
<br><br><br />
Results: All DNAs examined had a single BglII site, indicating that the pSB1C3-UAS DNA was successfully constructed.<br />
<br><br><br />
The BglII-digested pSB1C3-UAS DNA was purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
4. Digestion of pSB1C3 (PCR) with XbaⅠ and SpeⅠ<br />
<br><br />
PCR-amplified pSB1C3 DNA was double digested with XbaⅠ and SpeⅠ at 37℃ for 2 hours. <br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> XbaⅠ(NEB) </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.5uL </Td></Tr><Tr><br />
<Td> CIP </Td><Td> 0.5uL </Td></Tr><Tr><br />
<Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><Td> 3uL </Td></tr><br />
<td> Total </td><Td> 40uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were purified by QIA quick Gel Extraction Kit<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/da/0911kit.png" width="500" height="300"><br />
<br><br><br />
5. Insertion of GAL4, HS promoter or Act5C promoter-enhancer into the pSB1C3DNA<br />
<br><br />
<br><br />
Ligation reactions were carried out under conditions described below at 16℃ for 1hour .<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (vector) (digested with XbaⅠ and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (digestion with BglⅡ and SpeⅠ) </td><Td> 2.5uL </Td></tr><br />
<Tr><Td> HS promoter or Act5C promoter-enhancer (digested with XbaⅠ and BamHⅠ) </Td><Td> 2.5uL </Td></Tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCR products) (digested with XbaⅠ and SpeⅠ) </Td><Td> 2uL </Td></Tr><br />
<tr><td> GAL4 (digested with BglⅡ and SpeⅠ) </td><Td> 2.5uL </Td></tr><br />
<Tr><Td> HS promoter or Act5C promoter-enhancer (XbaⅠ and BamHⅠ) </Td><Td> 1.5uL </Td></Tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
13. Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(+) plate and cultured at 37℃for 16 hours.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<h2>September 12th</h2><br />
<br><br />
<strong>1-2-7 Purification of the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs</strong><br />
<br><br />
We purified the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs by QIA prep Spin Miniprep Kit from E. coli cultured in LB medium for 16 hours (9/11). <br />
<br><br><br />
<strong>1-1-47 Digestion of pSB1C3-UAS DNA by Spe1</strong><br />
<br><br />
We incubated BglⅡ-digested pSB1C3-UAS (9/11) DNA with SpeⅠfor 2 hours. The reaction conditions are shown below.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS-1, -2 </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3buffer(NEB) </td><Td> 5uL </Td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 1uL </Td></Tr><br />
<td> 100×BSA </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 3.5uL </Td></tr><br />
<td> Total </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
The digested DNA were applied to agarose gel electrophoresis. We isolated DNA from the gel by using QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of Agarose gel.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/a/ab/0912kit.png" width="500" height="300"><br />
<br><br><br />
<strong>1-1-48 and 2-2-6 Ligation</strong><br />
<br><br />
DNA fragment carrying EGFP or LacZ was ligated into the BglII and SpeI digested pSB1C3-UAS DNA .for 1hour at 16℃.<br />
<br><br><br />
Ligation reactions are listed below.<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3-UAS (double digested with BglII and SpeⅠ) </Td><Td> 1uL </Td></Tr><br />
<tr><td> EGFP fragments or LacZ fragments </td><Td> 2uL </Td></tr><br />
<Tr><Td> EGFP or LacZ </Td><Td> 2uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 2.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><Td> 2uL </Td></tr><tr><br />
<td> Total </td><Td> 7.5uL </Td></tr><br />
</Table><br />
<br><BR><br />
We ligated DNA fragment carrying GAL4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA for 1hour at 16℃.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (PCR) (double digested with XbaⅠand SpeI) </Td><Td> 2uL </Td></Tr><br />
<tr><td> GAL4(double digested with BglⅡ and SpeⅠ) </td><Td> 2uL </Td></tr><br />
<Tr><Td> HS fragments or Act5c fragments (double digested with XbaⅠand BamHⅠ) </Td><Td> 2uL </Td></Tr><tr><br />
<td> Ligation high </td><Td> 6uL </Td></tr><tr><br />
<td> Total </td><Td> 12uL </Td></tr><br />
</Table><br />
<br><BR><br />
<strong>1-1-49 and 2-2-7 Transformation of E. coli by Ligation products</strong><br />
<br><br />
We did transformation of E. coli Xl-1 blue with each of ligation products. Finally, we spread them on the LB Chloramphenicol(+) plate and incubated for 16 hours at 37℃.<br />
<br />
<br><br><br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week4p">>>>>>>>>>WEEK4</a></div><br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week2p
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2012-09-26T04:10:00Z
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<h2>August 31st</h2><br />
<br><br />
1. PCR amplification of DNA fragments containing UAS and Heat Shock promoter<br />
<br><br />
We have carried out the PCR reaction for UAS again. PCR amplification of Heat Shock promoter from pCasPer DNA was also carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 10uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 10uL </Td></Tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 3.2uL </Td></Tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<Tr><td> KOD plus </td><td> 2uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<Tr><td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature</Td><Td> Time </Td><Td>Cycle</Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
2. Purification of pAct5C DNA and pGaTB DNA<br />
<br><br />
pAct5C DNA and pGaTB DNA was purified from E. coli prepared on 8/29by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
<br />
<h2>September 1st</h2><br />
<br><br />
1. Agarose gel electrophoresis of PCR produsts and the purified pAct5C DNA and pGaTB DNA<br />
<br><br />
Agarose gel image is shown below. Left to right: HS promoter 10uL, UAS fragment 10uL, pAct5C DNA-1, pGaTB DNA-1, pGaTB DNA-2, pAct5C DNA<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/b/b0/0901akit.png" width="500" height="300"><br />
<br><br><br />
2. Expected bands for pAct5C DNA and pGaTB DNA were detected on the gel, but band for UAS fragment was not.<br />
<br><br><br />
<br />
<h2>September 3rd</h2><br />
<BR><br />
1. PCR amplification of DNA fragments containing GAL4, Act5C promoter and LacZ sequence<br />
<br><br />
pGaTBDNA was used as a template to amplify GAL4 sequence. pAct5C-LacZ DNA was used to amplify Act5C promoter-enhancer region and LacZ. <br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA template </td><td> 0.2uL </td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 5uL </td></tr><tr><br />
<td> 25mM MgSO<sub>4</sub> </td><td> 1.6uL </td></tr><tr><br />
<td> 10P 5’ primer </td><td> 1.5uL </td></tr><tr><br />
<td> 10P 3’ primer </td><td> 1.5uL </td></tr><tr><br />
<td> KOD plus </td><td> 1u L</td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 34.2uL </td></tr><tr><br />
<td> Total <td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Circle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 55℃(GAL4) and 58℃(Except for GAL4) </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min30sec </td><Td> 25 cycle </Td></tr><br />
<td> 68℃ </td><td> 2min30sec </td><Td></Td></tr><br />
<td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><br><br />
2. Agarose gel electrophoresis of the PCRproducts<br />
<br><br />
Left to right: 1kb ladder marker 5uL GAL4 fragments 10uL Act5C promoter-enhancer 10uL LacZ sequence <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/c/ca/0903kit.png" width="500" height="300"><br />
<br><br><br />
Results: Expected PCR products were all detected on the gel.<br />
<br><br />
These PCR products were purified by High Pure PCR Product Kit (Roche).<br />
<br><br><br />
<br />
<h2>September 4th</h2><br />
<br><br />
1. Digestion of GAL4 fragments by XbaⅠ and SpeⅠ<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 fragments prepared on 9/3 </Td><Td> 40uL </Td></Tr><br />
<tr><td> M buffer(TOYOBO) </td><td> 5uL </td></tr><br />
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> SpeⅠ(TOYOBO) </td><td> 0.5uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 4uL </td></tr><tr><br />
<td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
2. Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ<br />
<br><br />
pSB1C3 DNA prepared on 8/27, LacZ fragment prepared on 9/3 and EGFP fragments were digested with BglⅡ in the following conditions.<br />
<br><br />
<br><br />
3. Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ<br />
<br><br />
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> each DNA </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><td> 5uL </td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 4.5uL </td></tr><tr><br />
<td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ<br />
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.<br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 DNA </Td><Td> 23uL </Td></Tr><br />
<tr><td> M buffer(TOYOBO) </td><td> 10uL </td></tr><br />
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> SpeⅠ(TOYOBO) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> CIP </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><td> 64.5uL </td></tr><br />
<td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
4. Purification of GAL4 fragments<br />
<br><br />
After agarose gel electrophoresis, the digested GAL4 fragments were purified by QIA quick Gel Extraction Kit<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/b0/0904kit.png" width="500" height="300"><br />
<br><br><br />
5. Purification of BglII-digested LacZ fragments<br />
<br><br />
BglII-digested LacZ fragments were purified by High Pure PCR Product Kit.<br />
<br><br><br />
<br />
<h2>September 5th</h2><br />
<br><br />
1. Purification of EGFP fragments and HS promoter fragments<br />
<br><br />
EGFP fragments prepared on 8/29 and HS promoter fragments prepared on 8/31were purified by High Pure PCR Product Kit<br />
<br><br><br />
2. EGFP fragments and pSB1C3 DNA prepared on 8/27were digested with BglⅡ in the following reaction.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><td> EGFP </td><Td> pSB1C3 </Td></Tr><br />
<tr><td> DNA template </td><td> 40uL </td><Td> 23uL </Td></tr><br />
<Tr><Td> 3 buffer </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> BglⅡ </td><td> 0.5uL </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 4.5uL </td><Td> 21.5uL </Td></tr><br />
<td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
3. Purification of pSB1C3DNA digested with XbaⅠ and SpeⅠ<br />
<br><br />
Agarose gel electrophoresis image of the purified sample are shown below.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/3/3a/0905akit.png" width="500" height="300"><br />
<br><br><br />
4. SpeI digestion of the BglⅡ-digested LacZ, EGFP, pSB1C3 DNA<br />
<br><br />
SpeⅠ digestion was carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><td> 5uL </td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> CIP </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><td> 4uL </td></tr><br />
<td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
5. Ligation of pSB1C3 DNA and GAL4 fragment<br />
<br><br />
Ligation was carried out in the following reactions.<br />
<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (XbaⅠand SpeⅠdigested) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (XbaⅠ and SpeⅠ digested) </td><td> 1uL </td></tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3uL </Td></Tr><br />
<Tr><Td> Ligation high </Td><Td> 2.5uL </Td></Tr><br />
<td> Total </td><td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
6. The ligated products were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(+) plate<br />
<br><br><br />
7. Purification of pSB1C3, EGFP, LacZfragments double digested with BglⅡ and SpeⅠ<br />
<br><br />
The double digested samples were applied to the agarose gel electrophoresis and the DNA fragments were purified by QIA quick Gel Extraction Kit<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/63/0905bkit.png" width="500" height="300"><br />
<br><br><br />
<br />
<br />
<h2>September 6th</h2><br />
<br><br />
Transformation performed on September 5th was not successful, since we had no colony on the plate. <br />
<br><br><br />
1. PCR amplification of UAS , UAS-TNFAIP3, pSB1C3DNA<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.25uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 5uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 5uL </Td></Tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><tr><br />
<td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 34.15uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec</Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec</td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
2. PCR products were applied to the agarose gel electrophoresis<br />
<br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3<br />
<br><br><br />
<br />
3. Purifucation of pSB1C3 PCR products<br />
<br><br />
pSB1C3 PCR products were purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/5/53/0906kit.png" width="500" height="300"><br />
<br><br><br />
4. Ligation of pSB1C3 DNA and GAL4 fragment<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3(XbaⅠ and SpeⅠ cut) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> GAL4(XbaⅠ and SpeⅠ cut) </Td><Td> 1uL </Td></Tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 3uL </td></tr><br />
<Tr><Td> Ligation high </Td><Td> 2.5uL </Td></Tr><br />
<Tr><td> Total </td><td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
5. The ligation products were transformed into E. coli XL-1and spread on the LB Chloramphenicol(+) plate<br />
<br><br><br />
<br />
<br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week1p
Team:KIT-Kyoto/Notebook-week1p
2012-09-26T04:08:48Z
<p>Kazuko: </p>
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<h2>August 23rd</h2><br />
<br><br />
Transformation of E. coli with pEGFP-C2 DNA and pAct5C-GAL4 DNA<br />
<br><br />
E. coli transformed with pEGFP-C2 was spread on the LB Kanamycin(+) plate and that transformed with pAct5C GAL4 was spread on the LB ampicillin(+) plate.<br />
<br />
<br><br><br />
<br />
<br />
<h2>August 24th</h2><br />
<br><br />
The appropriate colonies were picked up and cultured in the LB medium containing the appropriate antibiotics.<br />
<br><br><br />
<br />
<br />
<h2>August 25th</h2><br />
<br><br />
1. Purification of the pEGFP-C2 DNA and the pAct5C-GAL4 DNA<br />
<br><br />
These plasmid DNAs were purified from E. coli by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
2. Digestion of pEGFP-C2 DNA with BamHⅠ and BglⅡt<br />
<br><br />
Restriction enzyme digestion was carried out in the following reaction<br />
<br><br><br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> All DNA </Td><Td> 500ng </Td><Td> 1ug </Td></Tr><br />
<tr><td> pEGFP-C </td><td> 3uL </td><Td> 6uL </Td></tr><br />
<Tr><Td> H buffer(TOYOBO) </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> BamHⅠ(TOYOBO) </td><td> 1uL </td><Td> 1uL </Td></tr><tr><br />
<td> BglⅡ(TOYOBO) </td><td> 1uL </td><Td> 1uL </Td></tr><tr><br />
<td> 100×BSA </td><td> 0.5uL </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 39.5uL </td><Td> 36.5uL </Td></tr><tr><br />
<td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><br><br />
<br />
3. Agarose gel electrophoresis of the digested DNA <br />
<br><br />
The digested DNA was applied to the agarose gel electrophoresis and linearized DNA was purified by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photograph of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d0/0825kit.png" width="500" height="300"><br />
<br><br><br />
4. Digestion of pSB1C3DNA with EcoRⅠ and SpeⅠ<br />
<br><br />
pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ at 37℃ for 16 hours.<br />
<br><BR><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA sample </td><td> 23uL </td></tr><br />
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr><br />
<tr><td> EcoRⅠ-HF(NEB) </td><td> 1uL </td></tr><tr><br />
<td> SpeⅠ(NEB) </td><td> 1.5uL </td></tr><br />
<td> 100×BSA </td><td> 0.5uL </td></tr><br />
<td> dH<sub>2</sub>O </td><td> 19uL </td></tr><br />
<td> Total <td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
<br />
5. PCR amplification of the UAS region and pSB1C3 DNA<br />
<br><br />
DNA fragments containing UAS was amplified from the pUAST DNA and the BglⅡ site-containing pSB1C3 was also amplified by PCR.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><Td> All samples </Td></Tr><br />
<tr><td> DNA template </td><td> 0.5uL </td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 10uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 10uL </td></tr><tr><br />
<td> 25mM MgSO4 </td><td> 3.2uL </td></tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<td> KOD plus </td><td> 2uL </td></tr><br />
<td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<td> Total <td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Circle </Td></Tr><br />
<tr><td> 95℃</td><td> 2min</td><Td></Td></tr><br />
<Tr><Td> 95℃</Td><Td> 15sec</Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃</td><td> 30min(UAS) or 2min10sec(pSB1C3)</td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃</td><td> 2min30sec</td><Td> 25 cycle </Td></tr><br />
<td> 68℃</td><td> 2min30sec</td><Td></Td></tr><br />
<td> 14℃</td><td> ∞</td><Td></Td></tr><br />
</Table><br />
<br><br><br />
<br />
<h2>August 26th</h2><br />
<br><br />
・Purification of the digested pSB1C3 <br />
<br><br />
pSB1C3 DNA double-digested with EcoRⅠ and SpeⅠ(prepared on 8/25) was applied to the agarose gel electrophoresis. The DNA fragment of interest was purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/0826.png" width="500" height="300"><br />
<br><br />
<br><br />
<h2>August 27th</h2><br />
<br><br />
1. Removing the multi-cloning site of the pEGFP-C2<br />
<br><br />
pEGFP-C2 DNA digested with BglⅡ and BamHⅠwas self ligated in the following reaction.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pEGFP-C2(cut) </Td><Td> 1uL </Td></Tr><br />
<tr><td> Ligation high </td><td> 2.5uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 5uL </td></tr><tr><br />
<td> Total <td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
2. The ligated products were transformed into the E. coli XL-1 blue and the E. coli was apreaded on the LB Kanamycin(+) plate and cultured at 37℃ for 16 hours.<br />
<br><br><br />
3. Agarose gel electrophoresis of the DNA fragments containing UAS and the PSR products from the pSB1C3 <br />
<br><br />
PCRproducts prepared on August 25th were applied to the agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/e/e6/0826bkit.png" width="500" height="300"><br />
<br><br><br />
Since the expected DNA fragments were detected, DNA was purified from the agarose gel by QIA quick Gel Extraction Kit. The purified DNAs were applied to the agarose gel electrophoresis as shown below.<br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/99/0826ckit.png" width="500" height="300"><br />
<br><br><br />
<br />
<h2>August 28th</h2><br />
<br><br />
1. Single colony isolation from the E. coli transformed with pEGFP-C2 DNA<br />
<br><br />
Single colony isolationwas carried out from the plate prepared on 8/27and cultured in 2.5mL LB Kanamycin(+) medium at 37℃ for 16 hours.<br />
<br><br><br />
2. Transformation of E. coli with pGaTB DNA or pAct5C-GAL4 DNA<br />
<br><br />
E. coli Xl-1 blue was transformed with pGaTBDNA or pAct5C-GAL4 DNA and cultured on LB ampicillin(+) plate for 16 hours at 37℃.<br />
<br><br><br />
<br />
<h2>August 29th</h2><br />
<br><br />
1. Single colony isolation from the E. coli transformed with pGaTB DNA or pAct5C-GAL4 DNA<br />
<br><br />
From the plate prepared on 8/28 single colonies were isolated and cultured in 2.5mL LB ampicillin(+) medium for 16 hours at 37℃.<br />
<br><br><br />
2. Purification of pEGFP-C2 DNA<br />
<br><br />
pEGFP-C2 DNA was purified from E. coli prepared on 8/28 by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
3. Cut check of the pEGFP-C2 DNA<br />
<br><br />
The purified pEGFP-C2 was digested with XhoⅠ and PstⅠ in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pEGFP-C2-1, -2, -3, -4 </Td><Td> 1uL </Td></Tr><br />
<tr><td> 10×H buffer(TOYOBO) </td><td> 0.5uL </td></tr><tr><br />
<tr><td> XhoⅠ </td><td> 0.1uL </td></tr><tr><br />
<tr><td> PstⅠ </td><td> 0.1uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 3.3uL </td></tr><tr><br />
<td> Total <td> 5uL </td></tr><br />
</Table><br />
<br><BR><br />
The digested samples were applied to the agarose gel electrophoresis.as shown below. <br />
<br><br><br />
Photo of the agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/97/0828kit.png" width="500" height="300"><br />
<br><br><br />
Results: The DNAs were undigested by these enzymes, confirming that the multi-cloning site of the pEGFP-C2 was successfully removed.<br />
<br><br><br />
4. PCR amplification of the DNA fragments containing UAS and EGFP<br />
<br><br />
Since we found that the previously used primers for PCR were wrong, PCR amplification of the DNA fragments containing UAS and EGFP region were carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><Td> All samples </Td></Tr><br />
<Tr><Td> DNA template </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 10uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 10uL </Td></Tr><br />
<Tr><Td> 25mM MgSO4 </Td><Td> 3.2uL </Td></Tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<Tr><td> KOD plus </td><td> 2uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<Tr><td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 1min(EGFP) </td><Td> 25 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 1min(EGFP) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
Agarose gel electrophoresis of the PCR products<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d2/0829b.png" width="500" height="300"><br />
<br><br><br />
Results: DNA fragments containing EGFP were successfully amplified, but not for the UAS.<br />
<br><br><br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week2p">>>>>>>>>>WEEK2</a></div><br />
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Kazuko
http://2012.igem.org/File:Side_partskit.jpg
File:Side partskit.jpg
2012-09-26T04:07:37Z
<p>Kazuko: </p>
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Kazuko
http://2012.igem.org/File:Side_tnfaip3kit.jpg
File:Side tnfaip3kit.jpg
2012-09-26T04:07:21Z
<p>Kazuko: </p>
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week3
Team:KIT-Kyoto/Notebook-week3
2012-09-26T03:52:15Z
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<td width="800px" valign="top"><div id="MIGI"><br />
<h2>August 20th</h2><br />
<br><br />
<br />
1. Isolation of DNA fragment from the gel<br />
Sample applied to the electrophoresis<br />
<br><br><br />
<strong>Composition</strong> <br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><Td> a product of PCR attB TNFAIP3(8/1) </Td></Tr><br />
<Tr><Td> DNA sample </Td><Td> 90μL</Td></Tr><br />
<Tr><Td> 6×Dye </Td><Td> 18μL</Td></Tr><br />
<Tr><Td> Total </Td><Td> 108μL</Td></Tr><br />
</Table><br />
<br><br />
<br />
<br><br />
<strong>Results</strong><br><br><br />
<img src="https://static.igem.org/mediawiki/2012/b/b8/0820.png" width="500" height="300"><br />
<br><br><br />
DNA was isolated from the agarose gel by QIA Quick Gel Extraction Kit, then the DNA was dissolved in 40 uL of TE.<br />
<br><br />
<br><br />
<br />
<h2>August 21st</h2><br />
<br><br />
<br />
1. Measuring the concentration of the attB TNFAIP3 DNA fragment<br />
<br><br />
The order of sample applied to the electrophoresis<br />
<br><br />
1kbmarker3uL,1uL of 5 fold dilution of attB TNFAIP3, 1uL of 10 fold dilution of attB TNFAIP3 DNA fragment,2uL of 1kbmarker, 1uL of 15 fold dilution of attB TNFAIP3 DNA fragment, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragment, 1kbmarker1uL, <br />
<br />
<br><br><br />
<strong>Result</strong><br />
<br><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/67/0821kit.png" width="500" height="300"><br />
<br><br><br />
The concentration of the isolated attB TNFAIP3 DNA fragments was estimated to be 35ng/uL.<br><br><br />
<br><br><br />
2. BP reaction<br><br />
1.5mL tube<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> attB TNFAIP3(35ng/μL)</Td><Td> 2μL </Td></Tr><br />
<Tr><Td> pDONR(150ng/μL) </Td><Td> 1μL </Td></Tr><br />
<Tr><Td> TE buffer </Td><Td> 5μL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 8μL </Td></Tr><br />
</Table><br />
<br><br><br />
We added 2uL of BP Clonase Ⅱ enzyme mix to this solution and incubate it for 2 hour. <br />
<br><br><br />
<br />
3. Transformation<br><br />
We added 100uL of XL1-Blue to the BP reaction products to do transformation.<br />
<br />
<br />
<br><br />
<br><br />
<br />
<h2>August 22nd</h2><br />
<br><br />
<br />
We isolated 6 colonies from a plate and incubated in 2.5mL of Kanamycin(+) LB liquid culture medium for 16 hours.<br />
<br />
<br />
<br><br><br />
<br />
<h2>August 23rd</h2><br />
<br><br />
1 Purification of the candidate pENTR-TNFAIP3 DNA<br />
<br><br />
We purified the candidate pENTR-TNFAIP3 DNA fro six independent colonies cultured on August 22 using QIA prep Spin Miniprep <br />
<br><br><br />
2 Characterization of the candidate pENTR-TNFAIP3 DNA<br />
<br><br />
We conducted PCR on the following condition to confirm the pENTR-TNFAIP3 DNA using primer designed for attB<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> BP TNFAIP3 </Td><Td> 0.2μL </Td></Tr><br />
<Tr><Td> 10× rTaq buffer </Td><Td> 0.8μL </Td></Tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 25mM MgCl<sub>2</sub> </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 10P 5'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> 10P 3'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> rTaq </Td><Td> 0.4μL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 13.4μL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 20μL </Td></Tr><br />
</Table><br />
<br><br><br />
Reaction<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> BP TNFAIP3 </Td><Td> 0.2μL </Td></Tr><br />
<Tr><Td> 10× rTaq buffer </Td><Td> 0.8μL </Td></Tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 25mM MgCl<sub>2</sub> </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 10P 5'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> 10P 3'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> rTaq </Td><Td> 0.4μL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 13.4μL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 20μL </Td></Tr><br />
</Table><br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> temperature </Td><Td> time </Td><Td> cycle </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 2min. </Td><Td> </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 15sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 60°C </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> </Td></Tr><br />
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td> </Td></Tr><br />
</Table><br />
<br><br />
<br><br />
The PCRproducts were applied to agarose gel electrophoresis <br />
<br><br />
From left to right: pDONR DNA 1uL, 6the candidate pENTR-TNFAIP3 2uL each<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/81/0823.png" width="500" height="300"><br />
<br><br />
<br><br />
The amplified DNA fragments were detected in the gel.<br />
<br><br><br />
3. LR reaction<br />
<br><br />
LR reactions were carried out under conditions as described below.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pENTR-TNFAIP3 (prepared on 8/23) </Td><Td> 1μL </Td></Tr><br />
<Tr><Td> pTFW or pTGW (Destination vectors) </Td><Td> 0.5μL </Td></Tr><br />
<Tr><Td> TE Buffer </Td><Td> 6.5μL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 8μL </Td></Tr><br />
</Table><br />
<br><br><br />
2uL LR clonaseⅡ enzyme mix was added to the reaction and incubated for 2.5 hour.<br />
<br><br><br />
4. The LR reaction products were transformed into E. coli XL1-Blue100uL according to the protocol and spread on the LB ampicillin(+) plate and cultured for 16 hours at 37℃.<br />
<br><br><br />
<br />
<h2>August 24th</h2><br />
<br><br />
Single colonies were isolated from a plate with the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 and cultured them in LB ampicillin(+) liquid medium for 16 hours at 37℃.<br />
<br><br><br />
<br />
<h2>August 25th</h2><br />
<br><br />
1. Purification of the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 DNA<br />
<br><br />
the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 were purified by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
2. Characterization of the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3<br />
<br><br />
We used the primer attB to confirm the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 for PCR reactions under conditions described below.<br />
<br><br><br />
Composition<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pTFW- or pTGW-TNFAIP3 </Td><Td> 0.2μL </Td></Tr><br />
<Tr><Td> 10× rTaq buffer </Td><Td> 0.8μL </Td></Tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 25mM MgCl<sub>2</sub> </Td><Td> 2μL </Td></Tr><br />
<Tr><Td> 10P 5'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> 10P 3'primer </Td><Td> 0.6μL </Td></Tr><br />
<Tr><Td> rTaq </Td><Td> 0.4μL </Td></Tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 13.4μL </Td></Tr><br />
<Tr><Td> Total </Td><Td> 20μL </Td></Tr><br />
</Table><br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> temperature </Td><Td> time </Td><Td> cycle </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 2min. </Td><Td> </Td></Tr><br />
<Tr><Td> 95°C </Td><Td> 15sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 60°C </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> 25cycle </Td></Tr><br />
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> </Td></Tr><br />
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td> </Td></Tr><br />
</Table><br />
<br><br />
<br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/0826.png" width="500" height="300"><br />
<br><br><br />
Results: Since the amplified DNAs with appropriate sizes were detected on the gel, pTFW-TNFAIP3 and pTGW-TNFAIP3 were appeared to be successfully constructed by the Gateway system ! At this point we renamed the pTFW-TNFAIP3 as pUAS-flag-TNFAIP3.<br />
<br><br><br />
<br />
<h2>August 26th</h2><br />
<br><br />
<br />
The female-virgin flies (w; Δ2,3) and male flies (yw) were collected for microinjection. The collected flies were kept in the 25℃ incubator separately.<br />
<br><br><br />
<br />
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Kazuko
http://2012.igem.org/File:0825kit.png
File:0825kit.png
2012-09-26T03:50:59Z
<p>Kazuko: uploaded a new version of &quot;File:0825kit.png&quot;</p>
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week4
Team:KIT-Kyoto/Notebook-week4
2012-09-26T03:43:05Z
<p>Kazuko: </p>
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<h2>August 27th</h2><br />
<br><br />
<br />
The female-virgin flies (w; Δ2,3) and male flies (yw) were collected for microinjection. The collected flies were kept in the 25℃ incubator separately.<br />
<br><br />
In total 50 pairs were collected.<br />
<br><br><br />
Large scale purification of the pUAS-flag-TNFAIP3 (pTFW-TNFAIP3) for microinjection into Drosophila embryos. <br />
<br><br />
To purify pUAS-flag-TNFAIP3 DNA, E. coli carrying the plasmid was cultured in 100 mL of LB ampicillin(+) nedium at 37℃ for 16 hours.<br />
<br><br><br />
<br />
<h2>August 28th</h2><br />
<br><br />
1.Sequencing of pUAS-flag-TNFAIP3 (pTFW-TNFAIP3) and pTGW-TNFAIP3 DNA<br />
<br><br />
To determine nucleotide sequence of pUAS-flag-TNFAIP3 (pTFW-TNFAIP3) and pTGW-TNFAIP3 DNA, we send the DNAs to the company. The sequencing data confirmed that pUAS-flag-TNFAIP3 is properly constructed.<br />
<br><br><br />
2. Midi-prep purification of the pUAS-flag-TNFAIP3 DNA<br />
<br><br />
The pUAS-flag-TNFAIP3 DNA was purified from E. coli cultured on on August 22 according to protocol using Pure LinkTM HiPure Plasmid Midiprep. Concentration of the obtained DNA was measured and adjusted it to be 1mg/mL<br />
<br><br><br />
<br />
<br />
<br />
<br />
<br />
<h2>August 30th</h2><br />
<br><br />
Micro-injection of the pUAS-flag-TNFAIP3 DNA into Drosophila embryos<br />
<br><br />
<strong>Microinjection</strong><br />
<br><br />
1. In the morning, w; Δ2,3 (female-virgin) were crossed with yw (male) in the cage and kept for 3 to 4 hours at 25℃.<br />
<br><br />
2. NaCl/Triton X-100, 10% Na-hypochloride, H<sub>2</sub>O were kept on ice.<br />
<br><br />
3. The tape was placed on the cover glass (3M tape, Cot No. W-18 pastes on the center).<br />
<br><br />
4. parafin oil is prepared.<br />
<br><br />
5. 1mg/ml DNA in microinjection buffere is prepared.<br />
<br><br />
6. glass needles for microinjection were prepared.<br />
<br><br />
7. The early embryos were collected on the nylon mesh and washed 3-4 times with NaCl/Triton X-100. Then embryos were dechorinated with 5 % Na-hypochloride. Then the dechorionated embryos were washed 7-8 times with H<sub>2</sub>O.<br />
<br><br />
8. The dechorinated embryos were placed on the prepared cover glass then parafin oil was dropped onto the embryos.<br />
<br><br />
9. DNA (pUAS-flag-TNFAIP3) was microinjected into embryos.<br />
<br><br />
10. After microinjection, the injected embryos were removed with the tape on the cover glass and placed onto the new egg plate. The tapes keep upside down from avoiding dry the embryos. <br />
<br><br />
11. The plate was kept in the 25℃ incubator.<br />
<br><br />
In total 692 embryos were microinjected with pUAS-flag-TNFAIP3 DNA (1mg/ml in microinjection buffer).<br />
<br><br />
Microinjection buffer: 5 mM KCl, 0.1 mM Sodium Phosphate, pH6.8<br />
<br><br />
<br />
DNA was EtOH-precipitated, washed with 75% EtOH and dissolved to 1 mg/ml in the Microinjection buffer.<br />
<br><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/d/d2/Injectionしてるとこ.JPG" width="500" height="300"><br />
<br><br><br />
<br />
<br />
<h2>August 31st</h2><br />
<br><br />
<br />
The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator.<br />
<br><br><br />
<br />
<br />
<br />
<h2>September 1st</h2><br />
<br><br />
The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator. About 20 larvae were put into a fly food vial.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<br />
<h2>September 2nd</h2><br />
<br><br />
The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator. In total 144 larvae were collected. The hatching efficiency after microinjection was calculated to be 20.8%.<br />
<br><br><br />
<br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Project
Team:KIT-Kyoto/Project
2012-09-26T03:11:53Z
<p>Kazuko: </p>
<hr />
<div>{{KIT-Kyoto.Header}}<br />
{{KIT-Kyoto}}<br />
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<div id="NAKAMI"><br />
<div id="Overview"><h2>Project Overview</h2><br />
<br>We try to develop new medicine for therapy of leukemia which is more free from side effects. <Br>For this study, we are going to use <I>Drosophila melanogaster</I>, a model organism to establish transgenic fly carrying responsible genes for human leukemia.<br />
</div><br />
<br><br />
<br><br />
<div id="Introduction"><h2>Introduction</h2><br />
<br><br />
<I>Drosophila melanogaster</I> has been used for a genetic study as model organism for a long time and brought us much discovery. And we are sure that the benefit continues from now on.<br />
<br><br />
Therefore we KIT-Kyoto team aim at the production of the disease model Drosophila which expresses the responsible gene of MALT lymphoma that is one of leukemia that we were not able to achieve in a meeting of the last year. It is thought that we can contribute to elucidation of the mechanism of this disease and the development of the therapeutic drug by promoting this project.<br />
<br><br />
In addition, we think about what we can do in order to continue researches using Drosophila melanogaster in the world. The structure and behavior of the gene cluster are often found by pushing forward the study about genes systematically and cooperatively.<br />
<br><br />
So, this year we aim at the design of the parts with which a study that we use the Drosophila can expand in iGEM in future.<br />
<br><br />
If these projects are realized, the study using D. melanogaster will step forward to the new one step again.<br />
</div><br />
<br><br />
<br><br />
<div id="Results"><br />
<h2>Results</h2><br />
<br><br />
<strong>BBa_K758006(UAS-EGFP)</strong><br />
<br><br><br />
We transfected this plasmid to Drosophila culture cells under two different conditions. We also transfected pAct5C-GAL4 plasmids which express GAL4 proteins in a culture cell line but not in other cell line.<br />
<br><br />
If the BBa_K758006 was assembled accurately, this plasmid expresses enough EGFP by activation of GAL4 protein. So the cell line that were co-transfected with pAct5C-GAL4 shows strong fluorescence, than the one without co-transfection with pAct5C-GAL4.<br />
<br><br><br />
Then we conducted experiment mentioned above. We made two groups of Drosophila culture cells. One group had been transfected with BBa_K758006 and pAct5C-GAL4, and the other one had been transfected with BBa_K758006 alone. And we calculated ratios between the number of green lighted cells to total cells about both groups.<br />
<br><br><br />
As results shown below, some green cells were detected among the cells co-transfected with both plasmids. But few green cells were detected among the cells transfected BBa_K758006 alone.<br />
<br><br />
And we also compiled statistics on the ratio of green lighted cells. In consequence, in the group transfected with pAct5C-GAL4, there were 45 green colored cells (12.6%) among 356 cells in total. And, without pAct5C-GAL4, there were only 2 cells were colored green (it is 0.5%) among 350 cells in total.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/f/f7/WIKI-Akit.png" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/c/c8/WIKI-Bkit.png" width="200" height="200"><br />
<br><br><br />
Cells transfected with BBa_K758006 and pAct5C-GAL4<br />
<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/6/63/WIKI-Ckit.png" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/3/31/WIKI-Dkit.png" width="200" height="200"><br />
<br><br><br />
Cells transfected with BBa_K758006 alone<br />
<br><br><br />
These results indicate that BBa_K758006 was activated by GAL4 protein and BB_K758006 EGFP expression was induced by GAL4 protein.<br />
<br><br />
In these circumstances, BBa_K758006 was working as expected.<br />
<br><br />
<br><br />
<br><br />
<br />
<br />
<br />
<strong>BBa_K758005(UAS-LacZ)</strong><br />
<br><br />
We transfected Drosophila cells with this plasmid, too. We devided the cells into two groups. One group had been co-transfected with pAct5C-GAL4 again, and the other group had been transfected with this plasmid alone. <br />
<br><br />
If BBa_K758005 was assembled accurately, the former group expresses LacZ strongly and the latter expresses weakly. <br />
<br><br><br />
Generally, to test the activation of LacZ, 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal) is used by mesuring blue color made by the degradation of X-Gal. However, in this experiment, we conducted immunostaining by red color to test the expression of LacZ (picture F, G, I, and J). Also we use 4', 6-diamidino-2-phenylindole (DAPI) that binds and emit blue light in order to count cells (picture E and H).<br />
<br><br><br />
As results shown below,some red cells were detected among the cells transfected with pAct5C-GAL4. They prove that LacZ is activated in this group. But the other group without transfection with pAct5C-GAL4 contains no red cells.<br />
<br><br />
We counted cells and calculated ratios of red cells to total cells between these two cases. In consequence, by the transfection of pAct5C-GAL4, 10.1%(42/415) cells showed red color. But, without pAct5C-GAL4, no cells showed red color (0/118 ).<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/b/bd/WIKI-Ekit.png" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/2/2d/WIKI-Fkit.png" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/2/2f/WIKI-Gkit.png" width="200" height="200"><br />
<br><br />
Drosophila cells transfected with BBa_K758005 and pAct5C-GAL4<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/5/53/WIKI-Hkit.png" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/1/16/WIKI-Ikit.png" width="200" height="200"> <img src="https://static.igem.org/mediawiki/2012/6/63/WIKI-Jkit.png" width="200" height="200"><br />
<br><br />
Drosophila cells transfected with BBa_K758005 alone<br />
<br><br />
<br><br />
These results show that BBa_K758005 was activated and expression of LacZ were induced in the presence of the GAL4 protein.<br />
<br><br><br />
Therfore, it can be said that BBa_K758005 was working as expected.<br />
<br><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<strong>Establishment of transgenic flies carrying pUAS-flag-TNFAIP3</strong><br />
<br><br><br />
We have injected 692 embryos (w-, delta2-3) with pUAS-flag-TNFAIP3 DNA (1mg/ml in microinjection buffer). Out of them 144 were hatched to larvae and 83 were further eclosed to adults. These 83 adult flies were crossed with yw flies and their progeny flies were inspected to identify successfully transformed w+ red eye flies. The red eye screening is still ongoing. However, we have identified six transformant strains that are listed below. Chromosomal linkage of the transgene is now under investigation.<br />
<br><br><br />
Strain 7, Strain 14, Strain 23, Strain 29, Strain 56, Strain 65, <br />
<br><br />
Strain 10<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/ba/HAEE.JPG" width="320" height="220"><br />
<br><br />
Strain 13<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/a/a6/Strain13.JPG" width="320" height="220"><br />
<br><br />
<br />
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Kazuko
http://2012.igem.org/Team:KIT-Kyoto/Notebook-week2p
Team:KIT-Kyoto/Notebook-week2p
2012-09-26T02:57:05Z
<p>Kazuko: </p>
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<div id="MIGI"><br />
<h2>August 31st</h2><br />
<br><br />
1. PCR amplification of DNA fragments containing UAS and Heat Shock promoter<br />
<br><br />
We have carried out the PCR reaction for UAS again. PCR amplification of Heat Shock promoter from pCasPer DNA was also carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 10uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 10uL </Td></Tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 3.2uL </Td></Tr><br />
<td> 10P 5’ primer </td><td> 3uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 3uL </td></tr><br />
<Tr><td> KOD plus </td><td> 2uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 68.3uL </td></tr><br />
<Tr><td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature</Td><Td> Time </Td><Td>Cycle</Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
2. Purification of pAct5C DNA and pGaTB DNA<br />
<br><br />
pAct5C DNA and pGaTB DNA was purified from E. coli prepared on 8/29by QIA prep Spin Miniprep Kit.<br />
<br><br><br />
<br />
<h2>September 1st</h2><br />
<br><br />
1. Agarose gel electrophoresis of PCR produsts and the purified pAct5C DNA and pGaTB DNA<br />
<br><br />
Agarose gel image is shown below. Left to right: HS promoter 10uL, UAS fragment 10uL, pAct5C DNA-1, pGaTB DNA-1, pGaTB DNA-2, pAct5C DNA<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/b/b0/0901akit.png" width="500" height="300"><br />
<br><br><br />
2. Expected bands for pAct5C DNA and pGaTB DNA were detected on the gel, but band for UAS fragment was not.<br />
<br><br><br />
<br />
<h2>September 3rd</h2><br />
<BR><br />
1. PCR amplification of DNA fragments containing GAL4, Act5C promoter and LacZ sequence<br />
<br><br />
pGaTBDNA was used as a template to amplify GAL4 sequence. pAct5C-LacZ DNA was used to amplify Act5C promoter-enhancer region and LacZ. <br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<tr><td> DNA template </td><td> 0.2uL </td></tr><br />
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr><br />
<tr><td> 2mM dNTPs </td><td> 5uL </td></tr><tr><br />
<td> 25mM MgSO<sub>4</sub> </td><td> 1.6uL </td></tr><tr><br />
<td> 10P 5’ primer </td><td> 1.5uL </td></tr><tr><br />
<td> 10P 3’ primer </td><td> 1.5uL </td></tr><tr><br />
<td> KOD plus </td><td> 1u L</td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 34.2uL </td></tr><tr><br />
<td> Total <td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Circle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr><br />
<tr><td> 55℃(GAL4) and 58℃(Except for GAL4) </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 2min30sec </td><Td> 25 cycle </Td></tr><br />
<td> 68℃ </td><td> 2min30sec </td><Td></Td></tr><br />
<td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><br><br />
2. Agarose gel electrophoresis of the PCRproducts<br />
<br><br />
Left to right: 1kb ladder marker 5uL GAL4 fragments 10uL Act5C promoter-enhancer 10uL LacZ sequence <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/c/ca/0903kit.png" width="500" height="300"><br />
<br><br><br />
Results: Expected PCR products were all detected on the gel.<br />
<br><br />
These PCR products were purified by High Pure PCR Product Kit (Roche).<br />
<br><br><br />
<br />
<h2>September 4th</h2><br />
<br><br />
1. Digestion of GAL4 fragments by XbaⅠ and SpeⅠ<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> GAL4 fragments prepared on 9/3 </Td><Td> 40uL </Td></Tr><br />
<tr><td> M buffer(TOYOBO) </td><td> 5uL </td></tr><br />
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 0.5uL </Td></Tr><br />
<tr><td> SpeⅠ(TOYOBO) </td><td> 0.5uL </td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 4uL </td></tr><tr><br />
<td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
2. Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ<br />
<br><br />
pSB1C3 DNA prepared on 8/27, LacZ fragment prepared on 9/3 and EGFP fragments were digested with BglⅡ in the following conditions.<br />
<br><br />
<br><br />
3. Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ<br />
<br><br />
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> each DNA </Td><Td> 40uL </Td></Tr><br />
<tr><td> 3 buffer(NEB) </td><td> 5uL </td></tr><br />
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 4.5uL </td></tr><tr><br />
<td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ<br />
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.<br />
<br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 DNA </Td><Td> 23uL </Td></Tr><br />
<tr><td> M buffer(TOYOBO) </td><td> 10uL </td></tr><br />
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> SpeⅠ(TOYOBO) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> CIP </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><td> 64.5uL </td></tr><br />
<td> Total </td><td> 100uL </td></tr><br />
</Table><br />
<br><BR><br />
4. Purification of GAL4 fragments<br />
<br><br />
After agarose gel electrophoresis, the digested GAL4 fragments were purified by QIA quick Gel Extraction Kit<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/b/b0/0904kit.png" width="500" height="300"><br />
<br><br><br />
5. Purification of BglII-digested LacZ fragments<br />
<br><br />
BglII-digested LacZ fragments were purified by High Pure PCR Product Kit.<br />
<br><br><br />
<br />
<h2>September 5th</h2><br />
<br><br />
1. Purification of EGFP fragments and HS promoter fragments<br />
<br><br />
EGFP fragments prepared on 8/29 and HS promoter fragments prepared on 8/31were purified by High Pure PCR Product Kit<br />
<br><br><br />
2. EGFP fragments and pSB1C3 DNA prepared on 8/27were digested with BglⅡ in the following reaction.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td></Td><td> EGFP </td><Td> pSB1C3 </Td></Tr><br />
<tr><td> DNA template </td><td> 40uL </td><Td> 23uL </Td></tr><br />
<Tr><Td> 3 buffer </Td><Td> 5uL </Td><Td> 5uL </Td></Tr><br />
<tr><td> BglⅡ </td><td> 0.5uL </td><Td> 0.5uL </Td></tr><tr><br />
<td> dH<sub>2</sub>O </td><td> 4.5uL </td><Td> 21.5uL </Td></tr><br />
<td> Total </td><td> 50uL </td><Td> 50uL </Td></tr><br />
</Table><br />
<br><BR><br />
3. Purification of pSB1C3DNA digested with XbaⅠ and SpeⅠ<br />
<br><br />
Agarose gel electrophoresis image of the purified sample are shown below.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/3/3a/0905akit.png" width="500" height="300"><br />
<br><br><br />
4. SpeI digestion of the BglⅡ-digested LacZ, EGFP, pSB1C3 DNA<br />
<br><br />
SpeⅠ digestion was carried out in the following reactions.<br />
<br><br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 40uL </Td></Tr><br />
<tr><td> 4 buffer(NEB) </td><td> 5uL </td></tr><br />
<Tr><Td> SpeⅠ </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr><br />
<Tr><Td> CIP </Td><Td> 0.5uL </Td></Tr><br />
<td> dH<sub>2</sub>O </td><td> 4uL </td></tr><br />
<td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
5. Ligation of pSB1C3 DNA and GAL4 fragment<br />
<br><br />
Ligation was carried out in the following reactions.<br />
<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3 (XbaⅠand SpeⅠdigested) </Td><Td> 1uL </Td></Tr><br />
<tr><td> GAL4 (XbaⅠ and SpeⅠ digested) </td><td> 1uL </td></tr><br />
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3uL </Td></Tr><br />
<Tr><Td> Ligation high </Td><Td> 2.5uL </Td></Tr><br />
<td> Total </td><td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
6. The ligated products were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(+) plate<br />
<br><br><br />
7. Purification of pSB1C3, EGFP, LacZfragments double digested with BglⅡ and SpeⅠ<br />
<br><br />
The double digested samples were applied to the agarose gel electrophoresis and the DNA fragments were purified by QIA quick Gel Extraction Kit<br />
<br><br><br />
Photo of agarose gel<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/6/63/0905bkit.png" width="500" height="300"><br />
<br><br><br />
<br />
<br />
<h2>September 6th</h2><br />
<br><br />
Transformation performed on September 5th was not successful, since we had no colony on the plate. <br />
<br><br><br />
1. PCR amplification of UAS , UAS-TNFAIP3, pSB1C3DNA<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> DNA template </Td><Td> 0.25uL </Td></Tr><br />
<tr><td> 10× KOD plus buffer </td><td> 5uL </td></tr><br />
<Tr><Td> 2mM dNTPs </Td><Td> 5uL </Td></Tr><br />
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><tr><br />
<td> 10P 5’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr><br />
<Tr><td> KOD plus </td><td> 1uL </td></tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 34.15uL </td></tr><br />
<Tr><td> Total </td><td> 50uL </td></tr><br />
</Table><br />
<br><BR><br />
Reaction conditions<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr><br />
<tr><td> 95℃ </td><td> 2min </td><Td></Td></tr><br />
<Tr><Td> 95℃ </Td><Td> 15sec</Td><Td> 30 cycle </Td></Tr><br />
<tr><td> 58℃ </td><td> 30sec</td><Td> 30 cycle </Td></tr><tr><br />
<td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr><br />
<tr><td> 68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr><br />
<tr><td> 14℃ </td><td> ∞ </td><Td></Td></tr><br />
</Table><br />
<br><BR><br />
2. PCR products were applied to the agarose gel electrophoresis<br />
<br><br />
From left to right: UAS, UAS-TNFAIP3, pSB1C3<br />
<br><br><br />
<br />
3. Purifucation of pSB1C3 PCR products<br />
<br><br />
pSB1C3 PCR products were purified from the gel by QIA quick Gel Extraction Kit.<br />
<br><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/5/53/0906kit.png" width="500" height="300"><br />
<br><br><br />
4. Ligation of pSB1C3 DNA and GAL4 fragment<br />
<br><br />
<Table Border Cellspacing="0"><br />
<Tr><Td> pSB1C3(XbaⅠ and SpeⅠ cut) </Td><Td> 1uL </Td></Tr><br />
<Tr><Td> GAL4(XbaⅠ and SpeⅠ cut) </Td><Td> 1uL </Td></Tr><br />
<Tr><td> dH<sub>2</sub>O </td><td> 3uL </td></tr><br />
<Tr><Td> Ligation high </Td><Td> 2.5uL </Td></Tr><br />
<Tr><td> Total </td><td> 7.5uL </td></tr><br />
</Table><br />
<br><BR><br />
5. The ligation products were transformed into E. coli XL-1and spread on the LB Chloramphenicol(+) plate<br />
<br><br><br />
<br />
<br />
<br />
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week3p">>>>>>>>>>WEEK3</a></div><br />
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Kazuko