Revision as of 13:45, 29 July 2010

Transformation of E. coli

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 minutes at 42°C.
Heat-shock the cells for 120 seconds, and place on ice again for 3-4 minutes.
Add 1 ml 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.
Incubate plates overnight at 37°C.

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 =Transformation of ''E. coli''=
+==Materials required==
+* Appropriate ''E. coli'' strains (200 µl)
+* Appropriate vector DNA
+* Heat block
+* Bucket of ice
+* Pipettes
+* Eppendorf tubes
+* 1.5% agar plate containing appropriate antibiotics
 ==Procedures==
-# 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.
+# 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 minutes 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 seconds, and place on ice again for 3-4 minutes.
-# Add 1ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking.
+# Add 1 ml 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.
 # Incubate plates overnight at 37°C.
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