Variable PCR results - Ideas and Suggestions?
Dr. Duncan Clark
duncan at genesys.demon.co.uk
Tue Oct 21 11:25:05 EST 1997
In article <344cac3d.2019757 at news.telenet.net>, Doug Jones
<jonesd at telenet.net> writes
>The PCR is done in a mix using 1.5 mM MgCl, 50mM KCl, 10mM Tris, 0.1%
>Triton X-100, .1% NP-40, .1% Gelatin, 0.25 mM each dNTP's, 5 U Taq
>(Promega) and primers in a volume of 50 microliters. The cycle is 1
>min at 95, 1 minute at 55, and 1.5 minutes at 72 degrees, for 35
>cycles in a Stratagene RoboCycler.
Why so much Taq. 'Normal' PCR with licensed Taq is 1.25u in 50ul. dNTPs
are normally 200uM. Any higher and they take out the MgCl2. It could be
that your 250uM dNTPs are dropping your Mg below the optimum for
consistent PCR. You are probably overkilling your annealing time and
could reduce that to 15secs. In a Light Cycler or Rapid Cycler the
denaturation and annealing times are less than 1sec! I'm not sure of
your extension time but the usual is 1min. per kb.
If you make up a master mix of say 5 reactions with all components
including template do they fail randomly or all work together. Could you
have a block problem ie cold spots in certain wells?
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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