reliable colony screening w/PCR?

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Tue Jun 3 00:04:43 EST 1997


In article <5mpohg$ev2$5 at cronkite.seas.gwu.edu>, sullivan at gwis2.circ.gwu.edu (Steven Sullivan) writes:
> Could someone post a reliable method (or Biotechniques citation for same)
> for rapid screening of bacterial colonies via PCR?  You know, one of those
> methods where you touch the colony with a toothpick or pipette tip and
> swirl it into the PCR reaction.  Thanks.
> 
> 

This one works consistently for me:

1) prepare a master mix ON ICE containing 0.2uM each vector primer
2.5U/100ul of taq and 200uM each dNTP. prepare enough for all the
plasmids you want analyzed.  I use 25ul/colony, but I have done it
with as little as 10ul/ colony.

2) quickly aliquot into individual PCR tubes so the mix has little or
no time to warm to RT. (important)

3) touck 10ul tip (the fine one) to a colony and dip into some LB or
onto a master plate then swish the tip into the mix on ice.  Avoid taking
any agar or LB into the mix as agar and NaCl from the LB can both inhibit
PCR.  Repeat for each colony.

4) cap each rxn with a drop of oil.  KEEP COLD on ice

5) Transfer the reactions DIRECTLY from ice into a PCR machine that
has already reached 95C.  Skip wells to minimize ramp times to 95C.

heat at 95C for 10 min
cycle 25 times with appropriate annealing temperatures 
and extension times for your primers and insert

Choice of vector primers is, of course, very important.  I don't know
which vectors you use, but I have some 24mers to the t7 and t3 of
bluescript that work well with a 65C anneal on a PE machine)

PCR buffer is not that important, but I usually use one that has 0.1%
triton in it.  Main thing is to keep everything cold so that primer
dimers are minimized (also going from ice directly to 95C)


	I have seen many variants on this  (i.e.don't use
toothpicks of a certain kind, boil and spin, etc in biotechniques and
posted here) but this was the simplest and most reliable in my hands.  
Hope you find sucess with it (or some other way) as well.

					Usual Disclaimers	SVEN



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