Team:Stockholm/17 September 2010
From 2010.igem.org
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+ | |} | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | == Mimmi == | ||
+ | |||
+ | === SOD / yCCS === | ||
+ | |||
+ | ==== over expression ==== | ||
+ | |||
+ | *Start culture | ||
+ | **10ml LB<sub>AMP</sub> + 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 | ||
+ | | rowspan="9" | [[Image:Place_for_picture.jpg|200px|thumb|left|]] | ||
+ | | rowspan="9" width="50" | | ||
+ | ! well | ||
+ | ! sample | ||
+ | | rowspan="9" | [[Image:Place_for_picture.jpg|200px|thumb|left|]] | ||
+ | |- | ||
+ | | 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 | ||
+ | | rowspan="8" width="100" | | ||
+ | ! Primers | ||
+ | | rowspan="8" width="100" | | ||
+ | ! colspan="2" | conditions | ||
+ | | rowspan="3" | | ||
+ | |- | ||
+ | | mastermix | ||
+ | | 24.5 | ||
+ | | rowspan="7" | | ||
+ | | pSB1_VF2 | ||
+ | ! time | ||
+ | ! °C | ||
+ | |- | ||
+ | | DNA | ||
+ | | 0.5 | ||
+ | | pSB1_VR | ||
+ | | 5m | ||
+ | | 95 | ||
+ | |- | ||
+ | | align="right" | tot | ||
+ | | 25µl | ||
+ | | rowspan="5" | | ||
+ | | 30s | ||
+ | | 95 | ||
+ | | ) | ||
+ | |- | ||
+ | | colspan="2" rowspan="4" | | ||
+ | | 30s | ||
+ | | 55 | ||
+ | | > 30 cycles | ||
+ | |- | ||
+ | | 1m20s | ||
+ | | 72 | ||
+ | | ) | ||
+ | |- | ||
+ | | 10m | ||
+ | | 72 | ||
+ | | rowspan="2" | | ||
+ | |- | ||
+ | | oo | ||
+ | | 25 | ||
|} | |} |
Revision as of 12:45, 4 October 2010
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 |