Two PCR quesions

Rajesh Kumar rk6n at DAYHOFF.MED.VIRGINIA.EDU
Mon Nov 4 11:37:16 EST 1996


 Dear Netters
         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?

With Thanks,
Rajesh Kumar





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