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Team:Brown/Notebook/October4
From 2010.igem.org
Monday, October 4, 2010
Digest and ligation of LacI into pSB1C3
LacI (linear, blunt ends) will be double digested with PstI and EcoRI and inserted into pSB1C3.
Digest
50ul total volume
- 25ul dH2O
- 5ul 10x buffer H
- 2.5ul EcoRI
- 2.5ul PstI
- 15ul DNA
Ran 2 tubes in parallel. the dH2O used for the first tube had a plastic-y smell, so the tube was discarded and this was noted in case of later error
Cleanup
Followed Qiagen PCR cleanup protocol
Ligation
30ul total volume
- 15ul 2x rapid buffer
- 2ul ligase (T4)
- 1ul vector
- 10ul insert (LacI)
- 2ul H2O
Digest AraC (2 Tubes)
50 μl total 25 μl H2O 5 μl 10x buffer H 2.5 μl EcoRI 25 μl Pst1 15 μl DNA (AraC-RFC25)
37°C at 21:36 out at 22:36 PCR purification, eluted in 30μl
Ligation 30 μl total 15 μl buffer 2x 2 μl ligase 2 μl vector (psB1C3) 11 μl insert (AraC)
in 4°C at 23:13
Nanodrop Results LacI - 147 ng/μl
Transformed Tat-L+pSB1C3 into XL1B (From 7/16)
- Used 10μl on a Chlor+ plate from 6/21
- No colonies seen after overnight at 37°C
