http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Kazuko&year=&month=
2012.igem.org - User contributions [en]
2024-03-29T05:53:39Z
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 />
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<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 />
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<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 />
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</td><br />
</tr><br />
<br />
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<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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</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 />
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{{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 />
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</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
2012-10-09T12:17:45Z
<p>Kazuko: </p>
<hr />
<div>{{KIT-Kyoto.Header}}<br />
{{KIT-Kyoto}}<br />
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<br><br />
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<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 />
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</td><br />
</tr><br />
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</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>
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<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 />
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<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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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 />
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background:transparent url(https://static.igem.org/mediawiki/2012/e/e2/Khaikei3.PNG);<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 />
<br />
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