Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Thu Nov 9 05:20:01 EST 2000
In article <firstname.lastname@example.org>, the eminent Philip
O'Brien at BIOSCI/MRC Human Genome Mapping Project Resource Centre wrote
>We have been getting a lot of smearing in our PCR reactions. Sometimes we
>get a discreet band, but most of the time its a smear. We get this with
>different primer pairs so it dosent't appear to be confined to one pair.
>The reactions are all set up on ice to minimize aberrant priming before
>thermalcycling. I would be grateful if anyone could give me information on
>how to eliminate the problem.
Too much enzyme - should be 1.25u per 50ulk but note a lot of unlicensed
enzyme may well be 5x that i.e they calim it is 5u/ul but it could
really be 20u/ul etc.
Too many cycles
Contaminated pipette barrels/water/buffer/dNTPs etc.
Go back to square one. Fresh barrier tips and bleached pipette barrels.
Take M13 universal and reverse sequencing primers and PCR 1ng for 15
cycles max of some simple small insert, maybe even just the polylinker,
in a pUC based plasmid. Use fresh reagents and if required run a
dilution series on the enzyme i.e. set up an initial 100ul PCR, add 2.5u
of enzyme, mix, remove 50ul to another 50ul PCR without enzyme, mix
repeat 4 or 5 times. Use 50C annealing. Does it work without smearing?
If OK move to genomic and try again.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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