Team:Newcastle/9 July 2010

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# Heat-shock the cells at 42°C for 120 secs, and place on ice again for 3-4 min.  
# 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).
# 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).
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# Plate out 200 µl/plate on LB (agar at 1.5%).
+
# 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)
#*10 LB plates were used (9 with ampicillin and 1 without ampicillin)
#**3 plates for 1:3
#**3 plates for 1:3

Revision as of 14:58, 21 July 2010

Contents

Transformation

Link to transformation protocol (to be added)

Aim:

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)


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 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.
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