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Team:Brown/Notebook/September22
From 2010.igem.org
(Difference between revisions)
(→Double digest of standard biobrick submission plasmid: psB1A3) |
(→Ligation of digested psB1A3 with Tat-L digest (from 9/16)) |
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Held at 4°C. | Held at 4°C. | ||
Then purified; ran PCR cleanup to purify, eluted in 50 μl EB. | Then purified; ran PCR cleanup to purify, eluted in 50 μl EB. | ||
| + | |||
| + | ===Ligation of digested psB1A3 with Tat-L digest (from 9/16)=== | ||
| + | (Using restriction digest enzymes EcoRI and Pst1) | ||
| + | 30 μl total rxn volume | ||
| + | *15 ul 2x rapid buffer | ||
| + | *2 ul ligase | ||
| + | *1 ul vector | ||
| + | *9 ul insert | ||
| + | *3 ul dH2O | ||
| + | |||
| + | ===Ligation of Tat-L into pGEM (using undigested Tat-L) PCR product=== | ||
| + | 30ul rxn total | ||
| + | *15 μl buffer 2x | ||
| + | *2 μl ligase | ||
| + | *1 μl pGEM | ||
| + | *9 μl insert | ||
| + | *3 μl dH2O | ||
| + | |||
| + | At 6:30 PM, both ligations put at 4°C. | ||
Latest revision as of 23:02, 23 October 2010
Wednesday, September 22, 2010
Double digest of standard biobrick submission plasmid: psB1A3
20 ul total 10μl H2O
- 2 μl 10x buffer H
- EcoRI 1 μl
- Pst1 μl
- 6 μl DNA (linear psB1A3)
In at 2 PM, 37°C thermocycler. Held at 4°C. Then purified; ran PCR cleanup to purify, eluted in 50 μl EB.
Ligation of digested psB1A3 with Tat-L digest (from 9/16)
(Using restriction digest enzymes EcoRI and Pst1) 30 μl total rxn volume
- 15 ul 2x rapid buffer
- 2 ul ligase
- 1 ul vector
- 9 ul insert
- 3 ul dH2O
Ligation of Tat-L into pGEM (using undigested Tat-L) PCR product
30ul rxn total
- 15 μl buffer 2x
- 2 μl ligase
- 1 μl pGEM
- 9 μl insert
- 3 μl dH2O
At 6:30 PM, both ligations put at 4°C.
