quick pcr (colony screening by PCR)
buratti at genes.icgeb.trieste.it
Thu Mar 17 08:43:08 EST 1994
frohlichm at STARBASE1.CALTECH.EDU ("FrohlichM") writes:
>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
What I do is to twirl the toothpick of cells in 10 microliters of water
placed at the bottom of a 0.5ml PCR tube and then add the PCR reaction mix
(ALWAYS do a positive and negative control as well). I then proceed with
the PCR reaction, taking care to heat all the samples for 2-3 minutes at
94 degrees before starting the programmed cycles. The godd thing about all
this is that instead of throwing away the toothpick I simply drop it in a
sterile tube with 2ml of TB. By the time the PCR has finished and the gel
is run you already have a fair amount of bacteria to work with. You'll
have to do minipreps of the positive clones to check that positives are
positives but I must say that it has always worked fine with me and that,
in this way, I've stopped spending my afternoons doing minipreps.
Hope this is of help too.
Emanuele Buratti, ICGEB, Trieste, Italy.
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