User:Daniel Goodman/Notebook/Cluzel/2010/03/31: Difference between revisions

Revision as of 12:10, 31 March 2010

Cluzel Lab Notebook
Daniel Goodman

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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)
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

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+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)
+*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
 |}
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