Team:KIT-Kyoto/kazukokekokko
From 2012.igem.org
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<h2>BP reaction by invitrogen gateway system</h2> | <h2>BP reaction by invitrogen gateway system</h2> | ||
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- | 1. PCR using | + | 1. PCR using primers containing the attB sequence. |
<br> | <br> | ||
- | + | 2.Purify PCR product. | |
- | + | ||
- | 2. | + | |
<br> | <br> | ||
3. Prepare vials in 1.5mL tube according to the following components. | 3. Prepare vials in 1.5mL tube according to the following components. | ||
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</Table> | </Table> | ||
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- | 4. | + | 4. Melt BP Clonase Ⅱ enzyme mix on ice. |
<br> | <br> | ||
- | 5. Add BP Clonase Ⅱ enzyme mix (2uL or 1uL) to | + | 5. Add BP Clonase Ⅱ enzyme mix (2uL or 1uL) to tube, vortex and spin them down. |
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6. Incubate vials at 25℃ for more than an hour. | 6. Incubate vials at 25℃ for more than an hour. | ||
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- | 7. Add Proteinase K | + | 7. Add Proteinase K solutionton (1uL) vials and vortex briefly. |
<br> | <br> | ||
8. Incubate at 37℃ for 10 minutes. | 8. Incubate at 37℃ for 10 minutes. | ||
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</Table> | </Table> | ||
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- | 2. | + | 2.Melt LR Clonase Ⅱ enzyme mix on ice |
<br> | <br> | ||
- | 3. Add LR Clonase Ⅱ enzyme mix to | + | 3. Add LR Clonase Ⅱ enzyme mix to tube (2uL or 1uL), votex and spin down. |
<br> | <br> | ||
4. Incubate vials at 25℃ for more than an hour. | 4. Incubate vials at 25℃ for more than an hour. | ||
<br> | <br> | ||
- | 5. Add Proteinase K solution to vials | + | 5. Add Proteinase K solution (1μl) to vials and vortex it briefly. |
<br> | <br> | ||
6. Incubate at 37℃ for 30 minutes. | 6. Incubate at 37℃ for 30 minutes. |
Revision as of 08:39, 16 September 2012
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BP reaction by invitrogen gateway system1. PCR using primers containing the attB sequence. 2.Purify PCR product. 3. Prepare vials in 1.5mL tube according to the following components.
4. Melt BP Clonase Ⅱ enzyme mix on ice. 5. Add BP Clonase Ⅱ enzyme mix (2uL or 1uL) to tube, vortex and spin them down. 6. Incubate vials at 25℃ for more than an hour. 7. Add Proteinase K solutionton (1uL) vials and vortex briefly. 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.Melt LR Clonase Ⅱ enzyme mix on ice 3. Add LR Clonase Ⅱ enzyme mix to tube (2uL or 1uL), votex and spin down. 4. Incubate vials at 25℃ for more than an hour. 5. Add Proteinase K solution (1μl) to vials and vortex it briefly. 6. Incubate at 37℃ for 30 minutes. |
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