Revision as of 14:41, 29 July 2010

Competent cells preparation

Inoculate 300 ml of LB broth in a conical flask and inoculate with 1/20 volume of an overnight culture of the desired strain.
Grow the cell at 37°C in an incubator(with a shaking platform so as to mix the media equally amongst the cells).
Chill cells by placing the flask on ice and harvest by centrifugation at 4°C for 10 minutes.
Resuspend the pellet in 100 ml of ice cold TFB1 and gently shake the tubes whilst placed on ice.
Repeat the the above mentioned step and carefully resuspend pellet in 20 ml ice cold TFB2.
Aliquot 200 µl volumes of the cell suspension into cold sterile microfuge (1.5 ml) tubes and flash freeze in dry-ice
Store at -80°C

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 ==Procedures==
-# Inoculate 300 ml of LB broth with 1/20 volume of an overnight culture of the desired strain.
+# Inoculate 300 ml of LB broth in a conical flask and inoculate with 1/20 volume of an overnight culture of the desired strain.
-# Grow the cell at 37°C (with shaking platform so as to mix the media equally amongst the cells)
+# Grow the cell at 37°C in an incubator(with a shaking platform so as to mix the media equally amongst the cells).
-# Next Chill cells on ice and harvest by centrifugation at 4°C for 10 minutes
+# Chill cells by placing the flask on ice and harvest by centrifugation at 4°C for 10 minutes.
-# Resuspend the pellet in 100 ml of ice cold TFBII
+# Resuspend the pellet in 100 ml of ice cold TFB1 and gently shake the tubes whilst placed on ice.
-# Aliquot 200 µl volumes of the cell suspension into cooled sterile microfuge tubes and flash freeze in dry-ice
+#Repeat the the above mentioned step and carefully resuspend pellet in 20 ml ice cold TFB2.
+# Aliquot 200 µl volumes of the cell suspension into cold sterile microfuge (1.5 ml) tubes and flash freeze in dry-ice
 # Store at -80°C
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