I'm having problems optimizing PCR primers for ChIP. They work great when I use genomic unsheared DNA as a template for the PCR reaction, however, that's not the case when I use 20% Input of sonicated DNA as a template. I don't think that it's a problem with the quality of sheared DNA as I have 2 primer pairs that always work and give me a great signal with both, unsheared genomic and sheared DNA. Is it just the case of bad primers even though they work very well on genomic DNA? I would greatly appreciate anyone's input on this as to how go about optimizing these primers. Thanks! Much appreciated!
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#2
KPDE
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Posted 07 November 2009 - 09:00 AM
pthomp, on Nov 5 2009, 02:13 PM, said:
I'm having problems optimizing PCR primers for ChIP. They work great when I use genomic unsheared DNA as a template for the PCR reaction, however, that's not the case when I use 20% Input of sonicated DNA as a template. I don't think that it's a problem with the quality of sheared DNA as I have 2 primer pairs that always work and give me a great signal with both, unsheared genomic and sheared DNA. Is it just the case of bad primers even though they work very well on genomic DNA? I would greatly appreciate anyone's input on this as to how go about optimizing these primers. Thanks! Much appreciated!
Sounds like there is a problem with the sheared DNA not the primers. How do you process the DNA for shearing and then crosslink reversal/proteinase K treatment?
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Posted 09 November 2009 - 02:17 AM
What´s the size of your amplicon?
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