Team:KIT-Kyoto/Notebook-week6

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             <div class="category"><img src="https://static.igem.org/mediawiki/2012/1/1f/Side_labnotekit.jpg" width="150" height="30"></div>
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<td width="800px" valign="top"><div id="MIGI">
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<h2>September 11th</h2>
<h2>September 11th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
 
-
<br><br>
 
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
<br><br>
<br><br>
-
<strong>Parts</strong>
 
-
<br><br>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td>pSB1C3-UAS-1, -2, -3, -4</Td><Td>5uL</Td></Tr>
 
-
<tr><td>3buffer(NEB)</td><Td>2uL</Td></tr>
 
-
<Tr><Td>BglⅡ</Td><Td>0.5uL</Td></Tr>
 
-
<td>dH<sub>2</sub>O</td><Td>12.5uL</Td></tr>
 
-
<td>Total</td><Td>20uL</Td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
<img src="https://static.igem.org/mediawiki/2012/0/0c/0911akit.png" width="500" height="300">
 
-
<br><br>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td>GAL4</Td><Td>40uL</Td></Tr>
 
-
<tr><td>4 buffer(NEB)</td><Td>5uL</Td></tr>
 
-
<Tr><Td>XbaⅠ(NEB)</Td><Td>0.5uL</Td></Tr>
 
-
<Tr><Td>SpeⅠ(NEB)</Td><Td>0.5uL</Td></Tr><Tr>
 
-
<Td>CIP</Td><Td>0.5uL</Td></Tr><Tr>
 
-
<Td>100×BSA</Td><Td>0.5uL</Td></Tr>
 
-
<td>dH<sub>2</sub>O</td><Td>3uL</Td></tr>
 
-
<td>Total</td><Td>40uL</Td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
<img src="https://static.igem.org/mediawiki/2012/d/da/0911kit.png" width="500" height="300">
 
-
<br><br>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td>1uL</Td></Tr>
 
-
<tr><td></td><Td>2.5uL</Td></tr>
 
-
<Tr><Td></Td><Td>2.5uL</Td></Tr>
 
-
<td>Ligation high</td><Td>6uL</Td></tr>
 
-
<td>Total</td><Td>12uL</Td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td>2uL</Td></Tr>
 
-
<tr><td></td><Td>2.5uL</Td></tr>
 
-
<Tr><Td></Td><Td>1.5uL</Td></Tr>
 
-
<td>Ligation high</td><Td>6uL</Td></tr>
 
-
<td>Total</td><Td>12uL</Td></tr>
 
-
</Table>
 
-
<br><BR>
 
<h2>September 12th</h2>
<h2>September 12th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
+
The transfected cells were examined under the confocal lazer microscope (Olympus, Fv10i)
 +
<br>
 +
Results: about 20 % cells were observed to be EGFP-positive, indicating that the P-element plasmid, pUAS-EGFP-TNFAIP3 is functional in Drosophila cells. We therefore decided to microinject this DNA into Drosophila embryos to establish transgenic flies.
<br><br>
<br><br>
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
<br><br>
<br><br>
-
 
-
<strong>Parts</strong>
 
-
<br><br>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td>pSB1C3-UAS-1, -2</Td><Td>40uL</Td></Tr>
 
-
<tr><td>3buffer(NEB)</td><Td>5uL</Td></tr>
 
-
<Tr><Td>SpeⅠ</Td><Td>1uL</Td></Tr>
 
-
<td>100×BSA</td><Td>0.5uL</Td></tr><tr>
 
-
<td>dH<sub>2</sub>O</td><Td>3.5uL</Td></tr>
 
-
<td>Total</td><Td>50uL</Td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
<img src="https://static.igem.org/mediawiki/2012/a/ab/0912kit.png" width="500" height="300">
 
-
<br><br>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td>1uL</Td></Tr>
 
-
<tr><td></td><Td>2uL</Td></tr>
 
-
<Tr><Td>EGFP or LacZ</Td><Td>2uL</Td></Tr>
 
-
<td>Ligation high</td><Td>2.5uL</Td></tr><tr>
 
-
<td>Total</td><Td>7.5uL</Td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td>2uL</Td></Tr>
 
-
<tr><td></td><Td>2uL</Td></tr>
 
-
<Tr><Td></Td><Td>2uL</Td></Tr>
 
-
<td>Ligation high</td><Td>6uL</Td></tr><tr>
 
-
<td>Total</td><Td>12uL</Td></tr>
 
-
</Table>
 
-
<br><BR>
 
<h2>September 13th</h2>
<h2>September 13th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
 
-
<br><br>
 
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
<br><br>
<br><br>
-
 
-
 
-
<strong>Parts</strong>
 
-
<br><br>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td>DNA sample</Td><Td>1uL</Td></Tr>
 
-
<tr><td>4 buffer(NEB)</td><Td>0.5uL</Td></tr>
 
-
<Tr><Td>EcoRⅠ-HF(NEB)</Td><Td>0.2uL</Td></Tr>
 
-
<Tr><Td>SpeⅠ(NEB)</Td><Td>0.2uL</Td></Tr><Tr>
 
-
<Td>100×BSA</Td><Td>0.05uL</Td></Tr>
 
-
<td>dH<sub>2</sub>O</td><Td>3.25uL</Td></tr>
 
-
<td>Total</td><Td>5uL</Td></tr>
 
-
</Table>
 
-
<BR><BR>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td>DNA sample</Td><Td>1uL</Td></Tr>
 
-
<tr><td>3 buffer(NEB)</td><Td>0.5uL</Td></tr>
 
-
<Tr><Td>BglⅡ</Td><Td>0.2uL</Td></Tr>
 
-
<td>dH<sub>2</sub>O</td><Td>3.3uL</Td></tr>
 
-
<td>Total</td><Td>5uL</Td></tr>
 
-
</Table>
 
-
<img src="https://static.igem.org/mediawiki/2012/3/3f/0913kit.png" width="500" height="300">
 
-
<br><br>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td>1uL</Td></Tr>
 
-
<tr><td></td><Td>1.5uL</Td></tr>
 
-
<Tr><Td>dH<sub>2</sub>O</Td><Td>2.5uL</Td></Tr>
 
-
<td>Ligation high</td><Td>2.5uL</Td></tr><tr>
 
-
<td>Total</td><Td>7.5uL</Td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td>2.5uL</Td></Tr>
 
-
<tr><td></td><Td>2.5uL</Td></tr>
 
-
<tr><td></td><Td>2uL</Td></tr>
 
-
<td>Ligation high</td><Td>7uL</Td></tr><tr>
 
-
<td>Total</td><Td>14uL</Td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td>1uL</Td></Tr>
 
-
<tr><td></td><Td>2uL</Td></tr>
 
-
<Tr><Td>dH<sub>2</sub>O</Td><Td>2uL</Td></Tr>
 
-
<td>Ligation high</td><Td>5uL</Td></tr><tr>
 
-
<td>Total</td><Td>10uL</Td></tr>
 
-
</Table>
 
-
<br><BR>
 
<h2>September 14th</h2>
<h2>September 14th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
 
-
<br><br>
 
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible. The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible. The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
<br><br>
<br><br>
-
 
-
 
-
<strong>Parts</strong>
 
-
<br><br>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td>DNA sample</Td><Td>1uL</Td></Tr>
 
-
<tr><td>4 buffer(NEB)</td><Td>1uL</Td></tr>
 
-
<Tr><Td>EcoRⅠ-HF(NEB)</Td><Td>0.2uL</Td></Tr>
 
-
<Tr><Td>SpeⅠ(NEB)</Td><Td>0.2uL</Td></Tr><Tr>
 
-
<Td>100×BSA</Td><Td>0.1uL</Td></Tr>
 
-
<td>dH<sub>2</sub>O</td><Td>7.5uL</Td></tr>
 
-
<td>Total</td><Td>10uL</Td></tr>
 
-
</Table>
 
-
<img src="https://static.igem.org/mediawiki/2012/e/ec/0914kit.png" width="500" height="300">
 
-
<br><br>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td>2uL</Td></Tr>
 
-
<tr><td></td><Td>3uL</Td></tr>
 
-
<tr><td></td><Td>1uL(HS) or 2.5uL(Act5c)</Td></tr>
 
-
<td>Ligation high</td><Td>6uL(HS) or 7.5uL(Act5c)</Td></tr><tr>
 
-
<td>Total</td><Td>12uL(HS) or 15uL(Act5c)</Td></tr>
 
-
</Table>
 
-
<br><BR>
 
<h2>September 15th</h2>
<h2>September 15th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
 
-
<br><br>
 
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
<br><br>
<br><br>
-
<strong>Parts</strong>
+
 
-
<br><br>
+
-
<Table Border Cellspacing="0">
+
-
<Tr><Td>DNA sample</Td><Td>1uL</Td></Tr>
+
-
<tr><td>4 buffer(NEB)</td><Td>1uL</Td></tr>
+
-
<Tr><Td>EcoRⅠ-HF(NEB)</Td><Td>0.2uL</Td></Tr>
+
-
<Tr><Td>SpeⅠ(NEB)</Td><Td>0.2uL</Td></Tr><Tr>
+
-
<Td>100×BSA</Td><Td>0.1uL</Td></Tr>
+
-
<td>dH<sub>2</sub>O</td><Td>7.5uL</Td></tr>
+
-
<td>Total</td><Td>10uL</Td></tr>
+
-
</Table>
+
-
<br><br>
+
-
<img src="https://static.igem.org/mediawiki/2012/d/d3/0915kit.png" width="500" height="300">
+
-
<br><br>
+
<h2>September 16th</h2>
<h2>September 16th</h2>
<BR>
<BR>
-
<strong>TNFA and API2</strong>
+
 
-
<br><br>
+
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. In total 83 flies from microinjected embryos were mated with yw flies. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible.
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. In total 83 flies from microinjected embryos were mated with yw flies. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible.
<br><br>
<br><br>
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<h2>September 17th</h2>
<h2>September 17th</h2>
<BR>
<BR>
-
<strong>TNFA and API2</strong>
+
 
-
<br><br>
+
The progeny flies were inspected by dissecting microscope to look for the successfully transformed w+ red eye flies (red eye screening). However, no red eye fly was found.
The progeny flies were inspected by dissecting microscope to look for the successfully transformed w+ red eye flies (red eye screening). However, no red eye fly was found.
<br><br>
<br><br>
-
 
+
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week7">>>>>>>>>>WEEK7</a></div>
-
<strong>Parts</strong>
+
-
<br><br>
+
-
<Table Border Cellspacing="0">
+
-
<Tr><Td>DNA sample</Td><Td>1uL</Td></Tr>
+
-
<tr><td>4 buffer(NEB)</td><Td>1uL</Td></tr>
+
-
<Tr><Td>EcoRⅠ-HF(NEB)</Td><Td>0.2uL</Td></Tr>
+
-
<Tr><Td>SpeⅠ(NEB)</Td><Td>0.2uL</Td></Tr><Tr>
+
-
<Td>100×BSA</Td><Td>0.1uL</Td></Tr>
+
-
<td>dH<sub>2</sub>O</td><Td>7.5uL</Td></tr>
+
-
<td>Total</td><Td>10uL</Td></tr>
+
-
</Table>
+
-
<br><br>
+
-
<img src="https://static.igem.org/mediawiki/2012/2/26/0917kit.png" width="500" height="300">
+
</div>
</div>
</div>
</div>

Latest revision as of 04:14, 26 September 2012

September 11th


The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

September 12th


The transfected cells were examined under the confocal lazer microscope (Olympus, Fv10i)
Results: about 20 % cells were observed to be EGFP-positive, indicating that the P-element plasmid, pUAS-EGFP-TNFAIP3 is functional in Drosophila cells. We therefore decided to microinject this DNA into Drosophila embryos to establish transgenic flies.

The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

September 13th


The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

September 14th


The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible. The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

September 15th


The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

September 16th


The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. In total 83 flies from microinjected embryos were mated with yw flies. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible.

September 17th


The progeny flies were inspected by dissecting microscope to look for the successfully transformed w+ red eye flies (red eye screening). However, no red eye fly was found.

Retrieved from "http://2012.igem.org/Team:KIT-Kyoto/Notebook-week6"