Problem with PCR

Varada P. Rao Memorial Univ. of Newfoundland St. John's Canada vprao at kean.ucs.mun.ca
Sat Dec 17 21:49:47 EST 1994


Hi netters,

I have experienced of late a new problem in addition to the weak signals 
that I often get in my PCR. I perform PCR to amplify T cell receptor cDNA
using several Vb-specific primers but using a single  constant region primer.
A year ago I have done initially some PCRs to deterimine optimal conditions
for my work. After that, I recently started again to repeat the previous 
observations.  All the reagents and primeres were stored in -20 Celcius.
Here comes the problem:

I do get the appropriate sized bands specifically ( negative controls do 
work) but now,  with  almost all  the appropriate  Vb-primers +  the same 
constant primer, I get " doublet-bands"; one of the correct size and
the second one is smaller by 50bp approx. This pattern is constant in every 
Vb-specific PCR.   
  
This double-band problem is never seen with beta-actin primers under 
identical conditions. 

I am really going mad with this. I would greatly  appreciate any input or
suggestions. 

Thanks very much in advance.

  vprao

"vprao at kean.ucs.mun.ca"
  



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