PCR problems

Sean David Moore smoore at mail2.sas.upenn.edu
Mon Oct 31 10:00:45 EST 1994


Make sure that you don't have too much starting RNA.  Also, make sure 
you're not saturating the second synthesis with 
product from the first.  If you have too much 
template, you will only get primer extension and not 
PCR.



Sean


mwatson at uniwa.uwa.edu.au wrote:

: 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

--
Sean Moore

VAMC Philadelphia,
Medical College of Pennsylvania,
and the University of Pennsylvania...

.

smoore at sas.upenn.edu





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