Latest revision as of 14:37, 26 October 2010

Transformation of E. coli

Thaw a 200 µl aliquot of E. coli DH4α and add the transforming DNA (10 ng of vector DNA in 10 µl).
Incubate on ice for 30 minutes.
Heat-shock the cells for 120 seconds at 42°C, and place on ice again for 3-4 minutes.
Add 1 ml of LB broth and incubate the cells at 37°C for 1-1.5 hr in a water bath with gentle shaking.
Plate 200 µl of transformed E. coli onto 1.5% agar plate containing the appropriate selection markers.
Incubate the plates overnight at 37°C.

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 ==Materials required==
-* Appropriate ''E. coli'' strains (200 µl)
+* ''E. coli'' DH5α (200 µl)
 * Appropriate vector DNA
 * Heat block
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 * 1.5% agar plate containing appropriate antibiotics
-==Procedures==
+==Protocol==
-# 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.
+# Thaw a 200 µl aliquot of ''E. coli'' DH4α and add the transforming DNA (10 ng of vector DNA in 10 µl).
-# Heat-shock the cells for 120 seconds, and place on ice again for 3-4 minutes.
+# Incubate on ice for 30 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.
+# Heat-shock the cells for 120 seconds at 42°C, and place on ice again for 3-4 minutes.
-# Plate out 100-200 µl/plate on LB (agar at 1.5%), containing the appropriate selection markers.
+# Add 1 ml of LB broth and incubate the cells at 37°C for 1-1.5 hr in a water bath with gentle shaking.
-# Incubate plates overnight at 37°C.
+# Plate 200 µl of transformed ''E. coli'' onto 1.5% agar plate containing the appropriate selection markers.
+# Incubate the plates overnight at 37°C.
+'''Go back to our [[Team:Newcastle/Protocol list|Protocol List]]'''
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