HELP with PCR

Sergio sergioal at bbm1.ucm.es
Mon Oct 22 12:23:39 EST 2001


I would say the smearing is due to one primer is annealing but the second one is
not. Try two test PCRs with only one of the primers each one, using same
conditions, and you probably will know which one is not annealing (no smear).
Check the primer or (maybe better) design a new one. Another posibility is to
reamplify that faint band you got in one of your PCRs.

Sergio
if everything fails, cursing is much better than smashing something, specially
if you are working in a foreing lab so nobody can understand what you say. It
was very useful when i went to Canada, at least for the first months. Afterwards
everybody in the lab learned the worst words of my lenguage.....

"Krzysztof W. Wasowicz" ha escrito:

> Hi,
> I have problem and I am getting desperate. I am trying to obtain a 220
> bp PCR product which is a fragment of bax cDNA. I am getting instead a
> smear starting from roughly 220 bp and running down to smaller
> molecules which ends around 145 bp. Sometimes (esp with lower
> annealing temps) I get a faint band at 220 bp. cDNA was prepared from
> total RNA obtained from skin and lymph node. I was trying to play with
> annealing temperature, but this smear is a consistent element of every
> PCR. When I rise the annealing temp it disappears, but the right band
> doesn't appear either. I test already second pair of primers. My
> primers have Tm of 62 and 64 centigrades. I perform 35 cycles (tried
> also 30 cycles w/o success), denaturation 95 cent 20 sec, annealing
> from 55 to 68 cent 20 sec, elong 72 1 min (tested from 10 sec to 1
> min). PCR machine Eppendorf Mastercycler Personal with heated lid. I
> do HotStart by assembling tubes on ice and putting them to pre-heated
> block.
> Any cues????????????? Help me or I will destroy the lab!!!!!!!!!!!!!!
> I already try to get that f..... PCR fragment for 2 months .
> Yours,
> Krzysztof W. Wasowicz
> Department of Animal Anatomy
> University of Warmia & Mazury
> ul. Oczapowskiego 13 (Bldg. 105J)
> 10-957 Olsztyn-Kortowo
> Poland
> Phone: +48-89-5233688
> Fax: +48-89-5234986
> e-mail: wasowicz at moskit.uwm.edu.pl
> ICQ: 58434323




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