Team:Paris Saclay/Notebook/August/22
From 2014.igem.org
Contents |
Friday 22nd August
Lab work
D - Lemon Scent
pPSI digestion
components | volumes |
---|---|
pPSI | 40μl |
FastDigest green buffer 10X | 5μl |
PacI | 2µl |
H2O | 2µl |
pPSI dephosphorylation
After 2 hours, add 1.5µL of Alkaline Phosphatase (AP) and let it 1 hour.
Then, inactivation of AP at 65°C during 15 minutes.
We've made a electrophoresis to check the digestion.
(photo)
Ligation of LS/GS/PS in pPSI
For each synthase, put:
components | volumes |
---|---|
synthase | 10μl |
pPSI | 2μl |
buffer | 2µl |
H2O | 5µl |
ligase | 1µl |
And let it in the freezer during 3 hours.
Transformation in competent E.coli DH5 Alpha
After 3 hours, take out the ligation tubes and add 100 µL of competent DH5 Alpha in each tube.
Then procede to the transformation protocol.
- 20 minutes at 4°C
- 2 minutes 30 seconds at 42°C
- 2 minutes at 4 °C
- Add 0.9 mL of LB into the tubes
And finally put it in a incubator at 37°C during 30 minutes