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Team:KIT-Kyoto/kazukokekokko
From 2012.igem.org
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<div id="MIGI"> | <div id="MIGI"> | ||
| - | BP reaction by invitrogen gateway system | + | <h2>BP reaction by invitrogen gateway system</h2> |
| + | <br> | ||
| + | 1. PCR using promers containing the attB sequence. | ||
| + | <br> | ||
| + | ＊attB sequence： | ||
| + | <br> | ||
| + | 2. Purification PCR products. | ||
| + | <br> | ||
| + | 3. Prepare vials in 1.5mL tube according to the following components. | ||
| + | <br><br> | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
<Tr><Td>attB DNA sample</Td><Td>75ng/reaction</Td></Tr> | <Tr><Td>attB DNA sample</Td><Td>75ng/reaction</Td></Tr> | ||
| Line 86: | Line 95: | ||
<Tr><Td>Total</Td><Td>8uL or 9uL</Td></Tr> | <Tr><Td>Total</Td><Td>8uL or 9uL</Td></Tr> | ||
</Table> | </Table> | ||
| - | |||
| - | |||
<br> | <br> | ||
| + | 4. Dissolve BP Clonase Ⅱ enzyme mix on ice. | ||
| + | <br> | ||
| + | 5. Add BP Clonase Ⅱ enzyme mix (2uL or 1uL) to vials, vortex and spin them down. | ||
| + | <br> | ||
| + | 6. Incubate vials at 25℃ for more than an hour. | ||
| + | <br> | ||
| + | 7. Add Proteinase K solutionto vials (1uL) and vortex shortly. | ||
| + | <br> | ||
| + | 8. Incubate at 37℃ for 10 minutes. | ||
| + | <br> | ||
| + | <br> | ||
| + | <h2>LR reaction by invitrogen gateway system</h2> | ||
| + | <br> | ||
| + | 1. Control vials in 1.5mL tube according to the following components. | ||
| + | <br><br> | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
<Tr><Td>BP DNA sample</Td><Td>50-150ng/reaction</Td></Tr> | <Tr><Td>BP DNA sample</Td><Td>50-150ng/reaction</Td></Tr> | ||
| Line 95: | Line 117: | ||
<Tr><Td>Total</Td><Td>8uL or 9uL</Td></Tr> | <Tr><Td>Total</Td><Td>8uL or 9uL</Td></Tr> | ||
</Table> | </Table> | ||
| + | <br> | ||
| + | 2. Dissolve LR Clonase Ⅱ enzyme mix on ice | ||
| + | <br> | ||
| + | 3. Add LR Clonase Ⅱ enzyme mix to vials by 2uL or 1uL, votex and spin down. | ||
| + | <br> | ||
| + | 4. Incubate vials at 25℃ for more than an hour. | ||
| + | <br> | ||
| + | 5. Add Proteinase K solution to vials by 1uL and vortex it shortly. | ||
| + | <br> | ||
| + | 6. Incubate at 37℃ for 30 minutes. | ||
<br> | <br> | ||
<br> | <br> | ||
| - | |||
| - | |||
| - | |||
</div> | </div> | ||
</td> | </td> | ||
Revision as of 14:49, 14 September 2012
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BP reaction by invitrogen gateway system1. PCR using promers containing the attB sequence. ＊attB sequence： 2. Purification PCR products. 3. Prepare vials in 1.5mL tube according to the following components.
4. Dissolve BP Clonase Ⅱ enzyme mix on ice. 5. Add BP Clonase Ⅱ enzyme mix (2uL or 1uL) to vials, vortex and spin them down. 6. Incubate vials at 25℃ for more than an hour. 7. Add Proteinase K solutionto vials (1uL) and vortex shortly. 8. Incubate at 37℃ for 10 minutes. LR reaction by invitrogen gateway system1. Control vials in 1.5mL tube according to the following components.
2. Dissolve LR Clonase Ⅱ enzyme mix on ice 3. Add LR Clonase Ⅱ enzyme mix to vials by 2uL or 1uL, votex and spin down. 4. Incubate vials at 25℃ for more than an hour. 5. Add Proteinase K solution to vials by 1uL and vortex it shortly. 6. Incubate at 37℃ for 30 minutes. |
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