Team:Stockholm/17 September 2010
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Contents |
Andreas
Plasmid preps
From 16/9 ON cultures
DNA concentration | ||
---|---|---|
Sample | Conc [ng/μl] | A260/A280 |
pSB1C3.N-TAT | 64.07 | n/a |
pSB1C3.N-Tra10 | 46.38 | n/a |
pSB1C3.N-LMWP | 65.77 | n/a |
pSB1K3.N-TAT⋅SOD⋅His 4 | 87.83 | n/a |
pSB1K3.N-TAT⋅SOD⋅His 5 | 8.464 | n/a |
pSB1K3.N-Tra10⋅SOD⋅His 5 | 6.872 | n/a |
pEX.SOD | 31.57 | n/a |
Mimmi
SOD / yCCS
over expression
- Start culture
- 10ml LBAMP + 100µl old culture (8:30)
- Measure OD=0.6 (11:30)
- At OD=0.6 add IPTG 1mM
- Take samples after:
- 0h
- 2h
- take 500µl, spinn down and remove LB
- resuspend in 50µl SDS buffer
- Heat in 95°C, 5min
- Freeze
- Re-heat in 95°C, 5min
- Run gel
PhastGel
well | sample | well | sample | |||
---|---|---|---|---|---|---|
1 | ladder | 1 | ladder | |||
2 | SOD.his 0h | 2 | yCCS 1 0h | |||
3 | SOD.his 2h | 3 | yCCS 1 2h | |||
4 | his.SOD 0h | 4 | yCCS 2 0h | |||
5 | his.SOD 2h | 5 | yCCS 2 2h | |||
6 | ladder | 6 | ladder |
his.SOD.cTAT
colony PCR
mix | (µl) | x8 | Primers | conditions | ||||
---|---|---|---|---|---|---|---|---|
mastermix | 24.5 | pSB1_VF2 | time | °C | ||||
DNA | 0.5 | pSB1_VR | 5m | 95 | ||||
tot | 25µl | 30s | 95 | ) | ||||
30s | 55 | > 30 cycles | ||||||
1m20s | 72 | ) | ||||||
10m | 72 | |||||||
oo | 25 |
Johan
PCR again, to see if the 900 nt-band or the 700 nt-band is correct
0,5 µl pol
0,5 µl dNTP
5 µl 5x buffer
1,5 µl for primer
1,5 µl rev primer
16 µl H2O
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