Team:Newcastle/9 July 2010

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Revision as of 14:29, 21 July 2010

Transformation

Link to transformation protocol (to be added)

Tubes used:

Each of the following five tubes contain 200 µl of competent E. coli DH5alpha. To this the DNA to be transformed was added.

1. 1:3 (contains LacI insert and pSB1AT3 vector in the proportion of 1:3)
2. 1:5 (contains LacI insert and pSB1AT3 vector in the proportion of 1:5)
3. Negative control for ligation (contains vector with no insert)
4. Control for transformation (without plasmid)
5. Control for transformation (with plasmid, pSB1AT3)

Protocol:

1. 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.
2. Incubate for 45 mins on ice.
3. Heat-shock the cells at 42°C for 120 secs, and place on ice again for 3-4 min.
4. 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).
5. Plate out 200 µl/plate on LB (agar at 1.5%), with ampicilin.
6. Incubate plates overnight at 37°C.