Team:KIT-Kyoto/kazukokekokko

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<div id="MIGI">
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<font color="#f5b1aa"><u>TNFAIP3</u></font>
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abstract
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<br>
 +
<font color="#f5b1aa"><u>PARTS</u></font>
 +
<br>
 +
<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.
 +
<br>
 +
2. Purify PCR product.
 +
<br>
 +
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>
<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.


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