"SMEAR" BUT NO BANDS WHEN DOING "LONG" PCR USING KLENTAQ1 WITH PFU
betinger at pobox.upenn.edu
Mon May 16 15:22:04 EST 1994
RE: "SMEAR" BUT NO BANDS WHEN DOING "LONG" PCR USING KLENTAQ1 WITH PFU
I am trying to PCR-amplify a stetch of genomic DNA cloned into a
cosmid. I do not know the size of the genomic DNA, since its genomic
structure is unknown. (Only the sequence of the cDNA is known, from which
I constructed 33mer primers for PCR). I guess that the size of the gene is
about 12kb. I used a 16:1 mixture of Klentaq1 (from AB Peptides, St.
Louis) and PFU DNA polymerase (Stratagene) in PC2 reaction buffer (AB
Peptides). I used 10pmol of each primer, 10 ng of cosmid and 1.2 ul of the
Klentaq1/PFU mixture. The reaction volume was 50ul. I used the Omnigene
Hybaid cycler with no initial denaturing step and then cycled with 2s at 95
degrees and annealing/extension temperature of 68-72 degrees for 24 minutes
for 24 cycles (according to Barnes' paper in PNAS in March '94).
Consistently, I get these surprising results when running out 25-50 ul on
an 0.8% agarose gel: Instead of discrete bands there is an intense
homogenous smear from the well down to the dye front , even in samples
without DNA template!
Does anyone have an explanation for this phenomenon and a suggestion how
to avoid it?
Thanks a lot!
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