Team:Newcastle/9 July 2010

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(Difference between revisions)
(→Transformation)
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Tubes used:
Tubes used:
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All tubes contain 200 µl of competent ''E. coli'' DH5alpha
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Each of the following tubes contain 200 µl of competent ''E. coli'' DH5alpha. To this the transforming DNA was added.
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# 1:3
+
# 1:3 (contains LacI insert and pSB1AT3 vector in the proportion of 1:3)
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# 1:5
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# 1:5 (contains LacI insert and pSB1AT3 vector in the proportion of 1:5)
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# Vector (no insert), negative control for ligation
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# Negative control for ligation (contains vector with no insert)
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# DH5alpha (without plasmid)
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# Control for transformation (without plasmid)
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# DH5alpha (with plasmid, pSB1AT3)
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# 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. 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. 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.
  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%), with ampicilin.
  6. Incubate plates overnight at 37°C.
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