RT-PCR Problems

gordon allison goa at aber.ac.uk
Sat Jul 6 04:45:32 EST 1996


In article <31DAB57F.536 at facstaff.wisc.edu>, pemozdzi at facstaff.wisc.edu 
says...
>
>I've solved my problem with multiple PCR Bands--BUT now I've got one 
>band at a larger number of bp than I should have--any ideas?

I assume your basing your product size on that you get with the positive 
control, right? Well on this basis you have no way of knowing what the 
product is, assuming you are using tissue and 'looking' for the RNA of 
interest. I was in a similar position and the only way of being sure is to 
get the product sequenced and screen a database. That will provide you with 
the evidence you need to answer your question. In addition, if you are 
using a two enzyme system (RT and DNA polymerase as separate enzymes) you 
can check for any DNA contamination you might have by excluding RT from the 
reaction - any DNA you have will then be amplified in the PCR cycling 
steps. 

Hope this helps. 

Jonathan Shillingford
jms93 at aber.ac.uk




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