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Team:Stockholm/21 July 2010
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(New page: {{Stockholm/Top2}} == Mimmi == === flourecent pEX === ==== colony PCR ==== {| ! Mix | (ml) | X8 | rowspan="6" witdh="100" | | |- | sH<sub>2</sub>O | 22.5 | 180 | rowspan="2" | *Take...)
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(New page: {{Stockholm/Top2}} == Mimmi == === flourecent pEX === ==== colony PCR ==== {| ! Mix | (ml) | X8 | rowspan="6" witdh="100" | | |- | sH<sub>2</sub>O | 22.5 | 180 | rowspan="2" | *Take...)
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Revision as of 20:11, 29 August 2010
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Contents |
Mimmi
flourecent pEX
colony PCR
| Mix | (ml) | X8 | ||
|---|---|---|---|---|
| sH2O | 22.5 | 180 | *Take two samples of respectively clone and add them to 10µl LB | |
| F primer | 1 | 8 | ||
| R primer | 1 | 8 | pEX.BBa_J18930 | |
| DNA | 0.5 | 4 | pEX.BBa_J18931 | |
| tot | 25µl | pEX.BBa_J18932 |
| Primers | ||||||
|---|---|---|---|---|---|---|
| pEX_VF | conditions | gel | ||||
| pEX_VR | time | °C | well | sample | ||
| 2m | 94 | 1 | ladder | |||
| 30s | 94 | ) | 2 | BBa_J18930A | ||
| 30s | 60 | > 30 cycles | 3 | BBa_J18930B | ||
| 2m | 72 | ) | 4 | BBa_J18931A | ||
| 10m | 72 | 5 | BBa_J18931B | |||
| oo | 10 | 6 | BBa_J18932A | |||
| 7 | BBa_J18932B | |||||
| 8 | control | |||||
| 9 | ladder | |||||
- Well 4 + 7 no product?
- Bad mixing?
over expression continue...
- Sonicate the samples on ice. 4X30s with 30s pause in between.
- Heat in 95°C for 1m
- Centrifuge 13000rpm for 1m
- Load 1µl on the comassie mini-gel with a special comb
--> Sample is slimey, DNA in it??
--> Hard to see the differences in the bands
LMWP
glycerol stock + preparation of plasmids
- Start cultures from the PCR colonies
| Mix | |
|---|---|
| LB | 12ml |
| Amp | 8µl |
| colony | 4µl |
- Keep in 30°C ON
