Cracking Procedure for Rapid analysis of Subclones (Chris Brown)
- 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)
- 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.
- Incubate at 70 C for 5 minutes. Cool to room temperature.
- Add 1.5ul 4M KCL vortex and place on ice 5 minutes.
- Microfuge for 3 minutes at 4 C.
- 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.