Freeze/Boil Cycling for Cell Lysis for rep-PCR

tromick at class.org tromick at class.org
Fri Feb 16 17:34:32 EST 1996


We use a simple boil prep for G- bacteria for PCR.  Simply grow your cells to 
max OD and spin down an Eppi full of cells.  Be sure to decant (by aspiration 
if possible) all spent medium as this will contain inhibitors to PCR.  If not 
sure, wash the cells once in water.  Then resuspend in 1/4 volume of water and 
boil 10 min.  Use 1-2 uL of this directly into the PCR reaction (50 uL), or 
freeze quickly (best at -80 C) until use.  The trick is to keep the prep either 
hot or cold to prevent nuclease action on the template.  Good luck.




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