quick pcr (colony screening by PCR)

FrohlichM frohlichm at STARBASE1.CALTECH.EDU
Wed Mar 16 19:18:52 EST 1994


DJP,

We screened colonies by PCR (but without ethidium bromide) using the
toothpick method of McCabe.  It was quick, easy and worked wonderfully well. 
Lift a colony with a steril toothpick; twirl the toothpick in 100 microliters
of TE with 1 percent triton-x 100 to get the cells off; heat to near boiling
for 5 min; spin down junk; use 5 microliters of supernatant for PCR under
your established conditions.  Running the products out on a gel doesn't take
that long, and allows checking the size of the product.

A shorter heating may work as well or better, and I've heard of people who
just add the toothpick of cells directly to the PCR reaction mix, though I've
not tried this.  

We typically put the plates back into 37 degrees for another half day to get
more growth so we had cells to pick from positive colonies.  

We used this PCR screening method in cloning a gene from Klebsiella:
Balakrishnan, R., M. W. Frohlich, P. T. Rahaim, K. Bachman & R. R. Yocum. 
1993.  J. Biological Chem. 268(33): 24792-24795

Good Luck!

Michael Frohlich



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