Help: Smear on RT-PCR

Madhu Singh msingh at gpu2.srv.ualberta.ca
Tue Oct 17 23:13:14 EST 1995


Hi experts:

I have recently started RT-PCR.  After reverse transcriptase reaction, I 
precipitated the products following phenol:chloroform extractions.  The 
amplification was done using standard protocol.  But, I found that there 
was a smear in test-samples only originating slightly below the wells.  
There was no sharp band seen.  The expected size of the amplified DNA was 
770 bp. But in all the samples, excepting the blank which contained 
everything except the template, an identical band of about 100 bp is 
seen.  The same template (RT product) was used for two different 
amplifications,both show this unexpected band.  I used NEB's Vent 
polymerase with proofreading activity.

Could someone explain what might have gone wrong?  Thanks in advance.


Madhuvrut

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email: msingh at gpu.srv.ualberta.ca
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