Team:Calgary/Notebook/Protocols/Process19

From 2011.igem.org

(Difference between revisions)
Line 1: Line 1:
{{Team:Calgary/Main_Header|notebook}}
{{Team:Calgary/Main_Header|notebook}}
{{Team:Calgary/Notebookbar|
{{Team:Calgary/Notebookbar|
-
TITLE=Making competent cells|
+
TITLE=Making Chemically Competent <i>E. coli</i>Cells|
BODY=<html>
BODY=<html>

Revision as of 03:24, 29 September 2011


Making Chemically Competent E. coliCells

This procedure was done using Top10 cells ordered from Invitrogen. 50 mL Falcon tubes were used for this protocol.

  1. Innoculate 5-10 mL LB at 37°C while shaking
  2. Subculture 1 mL of bacteria solution into 50 mL LB broth at 37° while shaking until OD600 is 0.4-0.6 (This step should require approximately 2.5 hours)
  3. Centrifuge the subculture at 10 000 rpm at 4°C for 2 minutes
  4. Resuspend pellet in 12.5 mL of cold CaCl2 (50 mM) and leave on ice for 10 minutes
  5. Centrifuge at 10 000 rpm at 4°C for 2 minutes and resuspend in 2 mL of cold CaCl2 (50 mM, 15% glycerol solution)
  6. Leave on ice for at least 30 minutes and then aliquot 200 uL and freeze at -80°C