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Team:Brown/Notebook/September22
From 2010.igem.org
(Difference between revisions)
(→Wednesday, September 22, 2010) |
(→Ligation of digested psB1A3 with Tat-L digest (from 9/16)) |
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===Ligation of digested psB1A3 with Tat-L digest (from 9/16)=== | ===Ligation of digested psB1A3 with Tat-L digest (from 9/16)=== | ||
(Using restriction digest enzymes EcoRI and Pst1) | (Using restriction digest enzymes EcoRI and Pst1) | ||
| - | 30 | + | 30 μl total rxn volume |
*15 ul 2x rapid buffer | *15 ul 2x rapid buffer | ||
*2 ul ligase | *2 ul ligase | ||
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.
