Team:Newcastle/9 July 2010

Transformation

Aims

The aim of this experiment is to transform competent E. coli DH5α 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 DH5α. 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

The transformation protocol was followed using the tubes above:

1. Thaw a 200 µl aliquot of E. coli DH5α 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).
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 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)
6. Incubate plates overnight at 37°C.

Results

Please see next day (link)