Team:KIT-Kyoto/kazukokekokko
From 2012.igem.org
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- | <h2>BP reaction by | + | <h2>BP reaction by Invitrogen gateway system</h2> |
<br> | <br> | ||
1. PCR using primers containing the attB sequence. | 1. PCR using primers containing the attB sequence. | ||
<br> | <br> | ||
- | 2.Purify PCR product. | + | 2. Purify PCR product. |
<br> | <br> | ||
- | 3. | + | 3. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix: |
<br><br> | <br><br> | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
- | <Tr><Td>attB | + | <Tr><Td>attB PCR product</Td><Td>75 ng/reaction (1-7 μL)</Td></Tr> |
- | <Tr><Td>pDONR vector</Td><Td>150ng/reaction</Td></Tr> | + | <Tr><Td>pDONR vector</Td><Td>150ng/reaction (1 μL)</Td></Tr> |
- | <Tr><Td>TE Buffer</Td><Td> | + | <Tr><Td>TE Buffer</Td><Td>to 8 μL</Td></Tr> |
- | + | ||
</Table> | </Table> | ||
<br> | <br> | ||
- | 4. | + | 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> | <br> | ||
- | 5. | + | 5. Incubate reaction at 25˚C for more than 1 hour. |
<br> | <br> | ||
- | 6. | + | 6. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly. |
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- | 7. | + | 7. Incubate at 37˚C for 10 minutes. |
<br> | <br> | ||
- | |||
<br> | <br> | ||
+ | <h2>LR reaction by Invitrogen gateway system</h2> | ||
<br> | <br> | ||
- | + | 1. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix: | |
- | + | ||
- | 1. | + | |
<br><br> | <br><br> | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
- | <Tr><Td> | + | <Tr><Td>Entry clones</Td><Td>50-150 ng/reaction (1-7 μL)</Td></Tr> |
- | <tr><td>Destination vector</td><td>150ng/reaction</td></tr> | + | <tr><td>Destination vector</td><td>150ng/reaction (1 μL)</td></tr> |
- | <Tr><Td>TE Buffer</Td><Td> | + | <Tr><Td>TE Buffer</Td><Td>to 8 − 9 μL</Td></Tr> |
- | + | ||
</Table> | </Table> | ||
<br> | <br> | ||
- | 2. | + | 2. Vortex LR ClonaseII enzyme mix briefly. Add 1 – 2 μL, to the components above and mix well by vortexing and spin down. |
<br> | <br> | ||
- | 3. | + | 3. Incubate reaction at 25˚C for 16 hours. |
<br> | <br> | ||
- | 4. | + | 4. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly. |
<br> | <br> | ||
- | |||
<br> | <br> | ||
- | + | 5. Incubate at 37˚C for 10 minutes. | |
<br> | <br> | ||
<br> | <br> |
Revision as of 02:26, 17 September 2012
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BP reaction by Invitrogen gateway system1. 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:
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 system1. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:
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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