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Team:Newcastle/9 July 2010
From 2010.igem.org
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Contents |
Transformation
Aims
The aim of this experiment is to transform competent E. coli DH5alpha with pSB1AT3 vectors containing the lacI insert (in the ratios of 1:3 and 1:5 of vector to insert)
Materials
Each of the following five tubes contain 200 µl of competent E. coli DH5alpha. To this the DNA to be transformed 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
The transformation protocol was followed using the tubes above:
- Thaw a 200 µl aliquot of E. coli DH5alpha and add the transforming DNA. For tubes 1-3 5 µl of vector was added, for tube 4 no vector was added and for tube 5 only 1 µl of vector was added. This is because tubes 1 to 3 have been ligated and will contain cells in different forms of the ligated vector whereas tube 5 contains the ligated vector (our control).
- 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 of solutiion from the 5 tubes above on LB plates (agar at 1.5%) using the glass balls.
- 10 LB plates were used (9 with ampicillin and 1 without ampicillin)
- 3 plates for 1:3
- 3 plates for 1:5
- Vector only plate (with no insert) i.e. 1:0
- 2 plates for without plasmid (one on LB with ampicillin and one without ampicillin)
- 1 plate with pSB1AT3 plasmid (only 1 µl of plasmid purified by SW and DY (198 ng/µl) was added)
- 10 LB plates were used (9 with ampicillin and 1 without ampicillin)
- Incubate plates overnight at 37°C.
Results
Please see next day (link)
   
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