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Cracking Procedure for Rapid analysis of Subclones (Chris Brown)

1. Pick individual colonies with sterile toothpicks and smear near the bottom of a microfuge tube. Add 50ul of 10 mM EDTA, pH 8.0. (put toothpick into identically numbered tube or grid plate to preserve colony)
   
   
2. Add 50ul of fresh Cracking buffer:
   
   2 ml 5M NaOH
   2.5 ml 10% SDS
   10 g Sucrose
   ddWater to 50 ml
   
   resuspend by vortexing.
   
   
3. Incubate at 70 C for 5 minutes. Cool to room temperature.
   
   
4. Add 1.5ul 4M KCL vortex and place on ice 5 minutes.
   
   
5. Microfuge for 3 minutes at 4 C.
   
   
6. Run 25-50ul on a 0.7 % gel. Don't forget uncut vector control. Look for an upward shift in electrophoretic mobility to indicate insert presence.