Team:Stockholm/13 September 2010
From 2010.igem.org
Contents |
Andreas
Preparation of Top10 chemically competent cells
None of the clones streaked onto the Amp 100 plate (11/9) grew, indicating no AmpR contamination in cells. One of the ON cultures were therefore selected for preparation of competent cells.
Procedures according to protocol. Growth conditions changed to 25 °C, 220 rpm (OD600 reached after ≈6 h).
A 100 μl aliquot was divided and spread onto Amp 100, Cm 25 and Km 50 plates to verify that they were not contaminated.
Cloning of N-CPPs into pSB1C3
Sequencing results from 8/9 returned.
- pSB1C3.nCPP 3 (failed)
- pSB1C3.nCPP 4 (fasta)
- pSB1C3.nCPP 6 (fasta)
- pSB1C3.nCPP 7 (fasta)
- pSB1C3.nCPP 8 (fasta)
Blastn alignments against N-Tra10, N-TAT and N-LMWP indicated successful cloning of N-Tra10 (clone 7) and N-TAT (clone 4).
Transformations
Since pSB1C3.N-TAT and pSB1C3.N-Tra10 colony samples were accidentally discarded, prepared plasmids were used to transform new cells in order to prepare glycerol stocks.
Standard transformation with 1 μl plasmid DNA.
- pSB1C3.N-TAT
- pSB1C3.N-Tra10
Sequencing
DNA concentrations of 11/9 plasmid preps were measured by Mimmi and samples were sent for sequencing for isolation of N-LMWP.
- pSB1C3.nCCP 2: ABS0045 B92
- pSB1C3.nCCP 3: ABS0045 B93
- pSB1C3.nCCP 5: ABS0045 B94
- pSB1C3.nCCP 8: ABS0045 B95
- pSB1C3.nCCP 9: ABS0045 B96
- pSB1C3.nCCP 10: ABS0045 B97
- pSB1C3.nCCP 11: ABS0045 B98
- pSB1C3.nCCP 12: ABS0045 B99
Mimmi
MITF-M
Site-Directed Mutagenesis control
mix | (µl) | Conditions | ||
---|---|---|---|---|
DNA | 20 | time | °C | |
10x buffer | 3 | 3h | 37 | |
sH2O | 5 | |||
XbaI | 1 | |||
AgeI | 1 | |||
tot | 30µl |
Gel
well | sample |
---|---|
1 | ladder |
2 | MITF-col 1 |
3 | MITF-col 1 cut X+A |
4 | MITF-col 2 |
5 | MITF-col 2 cut X+A |
6 | MITF-col 3 |
7 | MITF-col 3 cut X+A |
8 | MITF-col 4 |
9 | MITF-col 4 cut X+A |
10 | MITF-M |
11 | MITF-M cut X+A |
- Should have saved more original MITF (stupid!)
- Make new MITF-M
RBS
plasmid prep.
- Follow original protocol
- Wash 2 times with DNA wash buffer
- Eluate in 50µl
- Eluate 2 times with the same sH2O
DNA conc. RBS34a ~42ng/µl -> ~60ng/µl RBS34b ~41ng/µl -> ~60ng/µl