Team:Newcastle/9 July 2010
From 2010.igem.org
Transformation
- 1:3
- 1:5
- Vector (no insert), negative control for ligation
- DH5Alpha (without plasmid)
- DH5Alpha (with plasmid, pSB1AT3)
Protocol:
- Thaw a 200µl aliquot of E. coli DH5Alpha. Add the transforming DNA. We added 5µl of vectors from tubes 1 to 3 and 1µl of vector from tube 5 because vector from tube 5 is has not been ligated whereas tubes 1 to 3 have been ligated and will contain cells in different forms.
- Incubate for 45 min on ice.
- Heat-shock the cells at 42°C for 120 secs, and place on ice again for 3-4 min.
- Add 1ml of LB broth to each tube and incubate the cells at 37°C for 1.5 hr (static for initial 20 mins, shaking for remaining time).
- Plate out 200 µl/plate on LB (agar at 1.5%), with ampicilin.
- Incubate plates overnight at 37°C.