Help: PCR screen of colonies

rogier stugerNOvdSPAM at cellbiology.uni-frankfurt.de
Mon Sep 20 10:45:54 EST 1999


Hi,
this always worked for me:

E. coli: using a sterile yellow tip, pipet 100 ul sterile 
water into an eppie. then use this tip (which is still 
sterile) to pick a colony (take the entire colony!) and put 
it into the water.
vortex or pipet up and till the bacteria are suspended

use 3 ul as template in a standard 25 ul PCR (with 1 u of 
Taq. start with 5 min. at 95 C)

the E.coli suspension can be stored in the fridge for at 
least 2 months. Use 10 ul of the vortexed suspension to 
start a 25 ml culture for midiprepping


yeast: touch a colony with a tip, toothpick, whatever, and 
smear on a plate. then pick the entire colony, put into a 25 
uL PCR mix containing everything except polymerase. boil for 
10 minutes. add Taq and go PCR


good luck,
Rogier


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