TRUE postives or non specific bands, pls help

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kandyan
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Post by kandyan » Sat Jan 09, 2010 2:46 pm

Good one, thankx

clevermizo
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Post by clevermizo » Thu Jan 28, 2010 4:15 pm

I wouldn't believe anything on that gel as a positive. It looks like crap. You have to optimize PCR.

If the assay worked previously, look back and make sure you have the exact conditions. The most important thing in my experience when working with genomic DNA is the amount of template seed. I've used 400 ng genomic DNA template just to get PCR to work. But it works cleanly and perfectly. That's the first thing I'd optimize. In the context of a genome, the probability of your primers finding your template is lower than with sometime like a plasmid, with all the other DNA in the genome there and with all the possible secondary structure. To help out with secondary structure, try adjuvants in PCR (1-10% DMSO, for example). But the first thing to do is go back over your old notes concerning the assay at the time it was working and see what may be different between then and now.

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