Two PCR quesions
rk6n at DAYHOFF.MED.VIRGINIA.EDU
Mon Nov 4 11:37:16 EST 1996
I will appreciate if somebody there could explain me the
following two querries.
i) I prepared c-DNA from mouse mRNA using Not1-oligo dT primer.
I amplified it with a 5' primer specific to my sequence of interest
and the above oligo dT primer. I cut out a region from agarose gel
corresponding to the expected molecular weight of my sequence. I
reamplified it with a primer nested to my earlier 5'end primer and
the same oligo dT primer. I carried out control reactions simultaneously
with single primers. I got two products with good yield, but these were
also present in the reaction carried out with Not1-dT primer alone. The
annealing temp of PCR was equal to the Tm of the oligo dT primer. I am
not able to figure out how can oligo dT alone give such nice discrete
bands. I did this PCR reaction because I know only the 5' sequence of my gene.
ii) I am also getting PCR products using any single random primer with
above cDNA. Is it a normal thing in absence of a second primer?
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