quick pcr (colony screening by PCR)

Dave Dudley dudleyd at aa.wl.com
Thu Mar 17 20:37:06 EST 1994


> frohlichm at STARBASE1.CALTECH.EDU ("FrohlichM") writes:
> 
> >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
> 

I seem to keep deleting steps in this, but it still works....and I can't
imagine it being much simpler.

I take a 96-well plate, add 50 ul YT, LB (whatever is closest, but I
usually include 100 ug/ml AMP), to however many wells you wish to screen
colonies for.  Then pick colonies with a toothpick, and dip into one of the
wells.  I then set up PCR tubes with 45 ul of mix that contains 1 uM final
primers, dNTP, TAQ and buffer.  Add 5 ul 'bugs' from the 96-well plate, PCR
gem (or mineral oil) and start PCR.  Usually run 25 cycles, then run 10 ul
of the reaction on a gel and examine for bands.  The whole procedure takes
~4 hr.  Any positives can be picked from the 96-well plate to start
mini-preps and to streak a fresh agar plate.

-- 
Dave Dudley (dudleyd at aa.wl.com)
Dept. Signal Transduction
Parke-Davis
Ann Arbor, MI  USA



More information about the Methods mailing list