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5' RACE


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#1 MolEnt

MolEnt

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Posted 22 July 2010 - 08:43 AM

5 prime RACE has been giving me terrible problems lately.
I started by purifying total RNA, creating oligo-dT-primed cDNA with Thermoscript RT (Invitrogen), C-tailing with TdT for 30 minutes and then running a PCR reaction using the following cycle. My forward primer is GACATCGAAAGGGGGGGGGGG, so I used two annealing temps--one for the G-C bonds and a higer Tm for the entire primer.

94C 3 minutes (to activate Platinum Taq)

94C 30 sec
56C 30 sec
72C 1 min 20 sec
(5 cycles)

94C 30 sec
69C 30 sec
72C 1 min 20 sec
(40 cycles)

72C 10 min

I have designed three different reverse primers, based on a sequenced part of the gene, but none seem to work well. The best result looks like this, with non-specific bands. Any suggestions for how I can get good bands for sequencing?

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