Team:KIT-Kyoto/kazukokekokko

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<div id="MIGI">
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<font color="#f5b1aa"><u>TNFAIP3</u></font>
<br>
<br>
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  Drosophila melanogaster 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.
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<font color="#f5b1aa"><u>PARTS</u></font>
<br>
<br>
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  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.
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<h2>BP reaction by Invitrogen gateway system</h2>
<br>
<br>
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  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.
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1. PCR using primers containing the attB sequence.
<br>
<br>
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  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.
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2. Purify PCR product.
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<br>
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3. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:
 +
<br><br>
 +
<Table Border Cellspacing="0">
 +
<Tr><Td>attB PCR product</Td><Td>75 ng/reaction (1-7 μL)</Td></Tr>
 +
<Tr><Td>pDONR vector</Td><Td>150ng/reaction (1 μL)</Td></Tr>
 +
<Tr><Td>TE Buffer</Td><Td>to 8 μL</Td></Tr>
 +
</Table>
 +
<br>
 +
4. Vortex BP ClonaseII enzyme mix briefly. Add 1 - 2 μL to the components above and mix well by vortexing and spin them down.
 +
<br>
 +
5. Incubate reaction at 25˚C for more than 1 hour.
 +
<br>
 +
6. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.
 +
<br>
 +
7. Incubate at 37˚C for 10 minutes.
 +
<br>
 +
<br>
 +
<h2>LR reaction by Invitrogen gateway system</h2>
 +
<br>
 +
1. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:
 +
<br><br>
 +
<Table Border Cellspacing="0">
 +
<Tr><Td>Entry clones</Td><Td>50-150 ng/reaction (1-7 μL)</Td></Tr>
 +
<tr><td>Destination vector</td><td>150ng/reaction (1 μL)</td></tr>
 +
<Tr><Td>TE Buffer</Td><Td>to 8 − 9 μL</Td></Tr>
 +
</Table>
 +
<br>
 +
2. Vortex LR ClonaseII enzyme mix briefly. Add 1 – 2 μL, to the components above and mix well by vortexing and spin down.
 +
<br>
 +
3. Incubate reaction at 25˚C for 16 hours.
 +
<br>
 +
4. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.
 +
<br>
 +
<br>
 +
5. Incubate at 37˚C for 10 minutes.
<br>
<br>
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  If these projects are realized, the study using D. melanogaster will step forward to the new one step again.
 
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<div align="center">
<div align="center">
<strong>Our Sponsors</strong>
<strong>Our Sponsors</strong>
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<br><br>
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<a href="http://www.kit.ac.jp/"><img src="https://static.igem.org/mediawiki/2012/e/ea/KITkyoto.png" width="100" height="100"></a>
<br>
<br>
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Latest revision as of 12:49, 25 September 2012






TNFAIP3
PARTS

BP reaction by Invitrogen gateway system


1. PCR using primers containing the attB sequence.
2. Purify PCR product.
3. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:

attB PCR product75 ng/reaction (1-7 μL)
pDONR vector150ng/reaction (1 μL)
TE Bufferto 8 μL

4. Vortex BP ClonaseII enzyme mix briefly. Add 1 - 2 μL to the components above and mix well by vortexing and spin them down.
5. Incubate reaction at 25˚C for more than 1 hour.
6. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.
7. Incubate at 37˚C for 10 minutes.

LR reaction by Invitrogen gateway system


1. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:

Entry clones50-150 ng/reaction (1-7 μL)
Destination vector150ng/reaction (1 μL)
TE Bufferto 8 − 9 μL

2. Vortex LR ClonaseII enzyme mix briefly. Add 1 – 2 μL, to the components above and mix well by vortexing and spin down.
3. Incubate reaction at 25˚C for 16 hours.
4. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.

5. Incubate at 37˚C for 10 minutes.


Our Sponsors