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PCR advice

PCR advice - PCR - Polymerase Chain Reaction Forum

PCR advice - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 01-08-2010, 01:00 PM
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Default PCR advice



Hi,

I do a lot of PCR and my template is usually cDNA. I've recently started using genomic DNA and I can't even get the positive controls to work. I've used more than one primer set. Could anyone give me some tips on using genomic DNA? Annealing times, template concentration, etc. Any advice will be hugely appreciated.
I know there's a similar question on this forum but it's very specific to their work and I was hoping for more general advice.
Thanks.
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Old 01-12-2010, 03:50 PM
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Default Re: PCR advice

cDNA from RNA template? I would guess that your primer must contain exon sequence only thus in genomic DNA (gDNA) samples (both intron+exon). If was me I would BLAST the cDNA sequence against genomic DNA. Locate exact annealing position of the primer see whether it is present or not in gDNA.
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Old 01-22-2010, 10:23 PM
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Default Re: PCR advice

If you're using the same primers as you did with your cDNA template, then I agree with Butters that your primers may be crossing exon-junctions, hence won't prime gDNA.

When you say positive controls, do you mean the primers that come with a kit?

In general, I would set annealing temperature about 5-deg C above the Tm of the primers. Annealing time can be short, start with 10-seconds, then do about 1-min extension time per 1kb. For template concentration, 50 ng would be a safe low limit to use. More than 1ug would probably be overkill.

It's hard to give more advice without some specifics.
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Old 01-24-2010, 11:56 AM
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Default Re: PCR advice

Thanks everyone, I got it sorted. I used some different primers and it worked.
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