Team:Newcastle/9 July 2010

From 2010.igem.org

(Difference between revisions)
(→Transformation)
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===Transformation===
===Transformation===
 +
Tubes used:
 +
All tubes contain 200 µl of competent ''E. coli'' DH5alpha
# 1:3
# 1:3
# 1:5
# 1:5
# Vector (no insert), negative control for ligation
# Vector (no insert), negative control for ligation
-
# DH5Alpha (without plasmid)
+
# DH5alpha (without plasmid)
-
# DH5Alpha (with plasmid, pSB1AT3)
+
# DH5alpha (with plasmid, pSB1AT3)
Protocol:
Protocol:
-
# Thaw a 200µl aliquot of E. coli DH5Alpha. Add the transforming DNA. We added 5µl of vectors from tubes 1 to 3 and 1µl 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.
+
# 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.
-
# Incubate for 45 min on ice.  
+
# 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.  
# Heat-shock the cells at 42°C for 120 secs, and place on ice again for 3-4 min.  
-
# Add 1ml 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%), with ampicilin.  
# Plate out 200 µl/plate on LB (agar at 1.5%), with ampicilin.  
# Incubate plates overnight at 37°C.
# Incubate plates overnight at 37°C.

Revision as of 11:24, 9 July 2010

Transformation

Tubes used: All tubes contain 200 µl of competent E. coli DH5alpha

  1. 1:3
  2. 1:5
  3. Vector (no insert), negative control for ligation
  4. DH5alpha (without plasmid)
  5. DH5alpha (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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