Team:Newcastle/9 July 2010
From 2010.igem.org
Transformation
Tubes used: All tubes contain 200 µl of competent E. coli DH5alpha
- 1:3
- 1:5
- Vector (no insert), negative control for ligation
- DH5alpha (without plasmid)
- DH5alpha (with plasmid, pSB1AT3)
Protocol:
- Thaw a 200 µl aliquot of E. coli DH5alpha. Add the transforming DNA. We added 5 µl of vectors from tubes 1 to 3 and 1 µl 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 mins on ice.
- Heat-shock the cells at 42°C for 120 secs, and place on ice again for 3-4 min.
- Add 1 ml 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.