Team:Newcastle/9 July 2010
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Tubes used: | Tubes used: | ||
- | + | Each of the following tubes contain 200 µl of competent ''E. coli'' DH5alpha. To this the transforming DNA was added. | |
- | # 1:3 | + | # 1:3 (contains LacI insert and pSB1AT3 vector in the proportion of 1:3) |
- | # 1:5 | + | # 1:5 (contains LacI insert and pSB1AT3 vector in the proportion of 1:5) |
- | + | # Negative control for ligation (contains vector with no insert) | |
- | # | + | # Control for transformation (without plasmid) |
- | # | + | # Control for transformation (with plasmid, pSB1AT3) |
Protocol: | Protocol: |
Revision as of 11:33, 9 July 2010
Transformation
Tubes used: Each of the following tubes contain 200 µl of competent E. coli DH5alpha. To this the transforming DNA was added.
- 1:3 (contains LacI insert and pSB1AT3 vector in the proportion of 1:3)
- 1:5 (contains LacI insert and pSB1AT3 vector in the proportion of 1:5)
- Negative control for ligation (contains vector with no insert)
- Control for transformation (without plasmid)
- Control for transformation (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.