PCR problems

mwatson at uniwa.uwa.edu.au mwatson at uniwa.uwa.edu.au
Tue Oct 25 16:46:56 EST 1994


Dear Netters

I never thought I would see myself posting a PCR question but here 
goes!  I am performing RT PCR on Hepatitis C serum samples.  I 
extract the Viral RNA with a simple proteinase K/ phenol extraction. 
 I perform round one of my RT PCR in one tube (40 cycles with an 
initial 30 min incubation for thr RT) and get no visible band on the 
gel only a faint smere ranging from 0-about 1kb. I then use 1ul of 
this in a second round amplification with an inner primer set.  Upon 
running theses products on a gel I get the correct size band but also 
a huge amount of background smereing that ranges from 0 to 100kb+ 
(infact most of my amplification products remain in the well 
obviously too large to even run into the gel.  My question is where 
are these large PCR products comming from?  and how can I prevent it 
so that I can sequence my PCR products.  Changing the annealing up or 
down appears to have no effect other than to stop the reactions 
working completely.  Please help I am going nuts!

Thanks all

Mark Watson
p.s. please reply directly to my e-mail address



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