Team:KIT-Kyoto/kazukokekokko

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<A href="https://2012.igem.org/Team:KIT-Kyoto/Team-Kazuko" >Kazuko</A>
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<div id="MIGI">
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<font color="#f5b1aa"><u>TNFAIP3</u></font>
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<br>
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<font color="#f5b1aa"><u>PARTS</u></font>
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<br>
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<h2>BP reaction by Invitrogen gateway system</h2>
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<br>
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1. PCR using primers containing the attB sequence.
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<br>
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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:
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<br><br>
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<Table Border Cellspacing="0">
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<Tr><Td>attB PCR product</Td><Td>75 ng/reaction (1-7 μL)</Td></Tr>
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<Tr><Td>pDONR vector</Td><Td>150ng/reaction (1 μL)</Td></Tr>
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<Tr><Td>TE Buffer</Td><Td>to 8 μL</Td></Tr>
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</Table>
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<br>
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4. Vortex BP ClonaseII enzyme mix briefly. Add 1 - 2 μL to the components above and mix well by vortexing and spin them down.
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<br>
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5. Incubate reaction at 25˚C for more than 1 hour.
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<br>
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6. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.
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<br>
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7. Incubate at 37˚C for 10 minutes.
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<br>
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<br>
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<h2>LR reaction by Invitrogen gateway system</h2>
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<br>
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1. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:
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<br><br>
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<Table Border Cellspacing="0">
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<Tr><Td>Entry clones</Td><Td>50-150 ng/reaction (1-7 μL)</Td></Tr>
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<tr><td>Destination vector</td><td>150ng/reaction (1 μL)</td></tr>
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<Tr><Td>TE Buffer</Td><Td>to 8 − 9 μL</Td></Tr>
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</Table>
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<br>
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2. Vortex LR ClonaseII enzyme mix briefly. Add 1 – 2 μL, to the components above and mix well by vortexing and spin down.
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<br>
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3. Incubate reaction at 25˚C for 16 hours.
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<br>
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4. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.
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<br>
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<br>
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5. Incubate at 37˚C for 10 minutes.
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<br>
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<br>
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</div>
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<div id="NAKAMI">
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<div align="center">
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<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>
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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



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