Team:Newcastle/Transformation of E. coli

From 2010.igem.org

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===Protocol===
===Protocol===
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# Thaw a 200µl aliquot of the desired strain of ''E. coli'' and add the transforming DNA (10 ng of vector DNA in 10 µl). Incubate for 45 min at 42°C.
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# Thaw a 200 µl aliquot of the desired strain of ''E. coli'' and add the transforming DNA (10 ng of vector DNA in 10 µl). Incubate for 45 min at 42°C.
# Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
# Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
# Add 1ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking.
# Add 1ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking.
# Plate out 100-200 µl/plate on LB (agar at 1.5%), containing the appropriate selection markers.
# Plate out 100-200 µl/plate on LB (agar at 1.5%), containing the appropriate selection markers.
# Incubate plates overnight at 37°C.
# Incubate plates overnight at 37°C.

Revision as of 13:03, 28 July 2010

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Protocol

  1. Thaw a 200 µl aliquot of the desired strain of E. coli and add the transforming DNA (10 ng of vector DNA in 10 µl). Incubate for 45 min at 42°C.
  2. Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
  3. Add 1ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking.
  4. Plate out 100-200 µl/plate on LB (agar at 1.5%), containing the appropriate selection markers.
  5. Incubate plates overnight at 37°C.
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