RT-PCR non-specific bands

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Amtekoth
Amtekoth's picture
RT-PCR non-specific bands

Here's a question.

I'm getting multiple bands from RT-PCR on my gels. I'm using Invitrogen's Superscript one step kit with genomic RNA as my templates. I'm expecting one band in every lane, but I get a lot of minor bands too, some high, some low. For gel 2, I ran a subset where I raised my annealing temps (if I go 2 more degrees higher everything goes away), my extension temps, and my RT temp. Since I'm expecting a 300-400 bp amplicon, I also cut down the time for my DNA extensions to get rid of the big non-specific bands.

Any advice?

Thanks,

Ed

Fraser Moss
Fraser Moss's picture
Try touchdown PCR

Try touchdown PCR

This is when you start with high annelaing temperatures and decrease it with every other cycle by 0.5 or 1 degree C.

www.pcrlinks.com/variants/touchdown.htm

http://en.wikipedia.org/wiki/Touchdown_PCR

http://www1.qiagen.com/faq/faqview.aspx?faqid=75&SearchText=&FaqCategoryId=0&MenuItemId=0&catalog=1&ProductLineId=1000097

'Touchdown' PCR to circumvent spurious priming during gene amplification.
Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS.

Centre for Molecular Biology and Biotechnology, University of Queensland, Brisbane, Australia.

PMID: 1861999 [PubMed - indexed for MEDLINE]

You will get a greater yield of specific band over any mis-primed amplicons.

Do any of your lanes contain a no template control? This should always be done to check for primer or other contamination.

Amtekoth
Amtekoth's picture
Thanks, I'll give that a shot

Thanks, I'll give that a shot. We usually use an old Perkin Elmer, which has an awful programming interface IMO, making touchdown a pain to set up. I'll probably go over to campus and run some Q-RT-PCR on an iCycler and see if that works.

Thanks,

Ed