Team:Newcastle/9 July 2010

From 2010.igem.org

(Difference between revisions)
(Protocol:)
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===Transformation===
===Transformation===
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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)
Link to transformation protocol (to be added)
Link to transformation protocol (to be added)
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# 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).
# Plate out 200 µl/plate on LB (agar at 1.5%).
# Plate out 200 µl/plate on LB (agar at 1.5%).
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#*  
+
#*
# Incubate plates overnight at 37°C.
# Incubate plates overnight at 37°C.

Revision as of 14:46, 21 July 2010

Transformation

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) 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 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/plate on LB (agar at 1.5%).
  6. Incubate plates overnight at 37°C.