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Ask for help w/ 3' RACE


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#1 jiang21

jiang21

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Posted 07 August 2010 - 08:23 PM

I have worked on 3' RACE for one gene for a couple of months. After RACE w/ Invitrogen kit, I tried several taqs from Invitrogen, Bioline, Strategen. I designed 6 gene specific primers (25-28 in length) for 3'. Because of many repeats @ 3' region, I have to design primers avoiding those regions - so expected products should be all > 1k bp.

I tried to optimize PCR conditions (AT, Mg, elonging time...). But each time I ended up w/ nothing (or primer dimers) or small mismatched bands (sequenced). Pretty frustrated!!! (Another gene 3' RACE worked so I think the RACE reation is fine. )

Would anyone please give me any suggestions or what kind of Taq you used or PCR conditions?? Thanks a lot!




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