Quick PCR

Bob DeLisio delisior at rnisd0.DNET.roche.com
Thu Apr 22 15:36:25 EST 1993


Hi Pat:

I am posting this on the net because I think others might find it useful.

PCR COLONY MICROSCREEN

1. Pick colonies into 100 microliters of LB+antibiotic in a sterile 
	microtiter well.
2. Grow 2-4 hours at 37 C on shaker platform until turbid.
3. Add 2 microliters of the culture to 23 microliters of PCR reaction mix*.
4. PCR cycles:

	Denature	96 C for 6 min.
	
	25 Cycles of: 	96 C x 30 sec.
		     	50 C x 30 sec.
		     	72 C x 90 sec.

	Extend		72 C x 10 min.

5. Run 5 microliters of the reaction on a minigel.

*PCR reaction mix
per 2 microliters of culture add:

2.5 microliters 10x PCR buffer
17.5 microliters ddW
1 microliter dNTP mix**
0.125 microliters Taq polymerase
1 microliter (20pM) primer #1
1 microliter (20pM) primer #2

**this is made by diluting 10 mM stocks to 1.25 mM.
(e.g. 12.5 microliters each dNTP plus 50 microliters ddW).

Regards
Bob DeLisio
Roche Institute of Molecular Biology
Roche Research Center
Nutley, NJ





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