User:Daniel Goodman/Notebook/Cluzel/2010/03/31

Protocol for Competent Cells

 * Grow 250 ml flask, one colony, 50 ml LB at 37 deg for 4 hours (OD must be 0.3-0.4) in shaker (check at ~7pm) - OD was 0.83


 * Spin down 20 mls in 2 50 ml epis at 4 degrees in centrifuge
 * 3200 RCF (4000 RPM) 10 minutes 4 degrees
 * pour of supernatant, add 40 mls 4 deg filtered water each (maybe add 5 first, vortex, add 35 more)
 * make sure pellet is dissolved
 * repeat above 2x
 * resuspend pellets in 10% glycerol (5 mls each)
 * spin, remove glycerol supernatant
 * resuspend in 0.5 ml 10% glycerol each, combine tubes
 * make 20x 50 ul aliquots, put in -80 freezer

Agarose Chip Prep

 * Put silicon chips in acetone (under fume hood) in glass flask for 5 minutes, heated slightly
 * Made 3ml 5% agarose, heat to 80 deg
 * Using stationary phase GFP cells, dilute a few colonies in DI water to OD 0.05.


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