Quick PCR

[Mike Morris]

In article <9304222010.AA09552 at mailgate.roche.com>, delisior at rnisd0.DNET.roche.com (Bob DeLisio) writes:
> 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).
> 

There is a much quicker way of doing this: 

	pick the colony to 200 ul TE, with a yellow tip
	stir
	streak on agar as a stock
	BOIL the bugs/TE, about 5 mins
	spin 2 min
	take 2 ul of this to start your normal PCR.

	Works every time, and is _quick_



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??         Mike Morris                 ??
??         Medical Genetics            ??
??         Geneva                      ??
??         Switzerland                 ??
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