Designation of PCR primers in ChIP experiment - (Sep/28/2007 )

Hello everyone!
I have been doing ChIP experiment for two months, I want to know whether there are bindings between estrogen receptor and some specific sites of a gene.
The problem is about ChIP PCR, I can not get single band in target site, too more non-specific bands or no band at all. I have designed 4 pairs of primers in one site, even though, I can not get any specific PCR result which can be used to perform ChIP.
The length of every primer is about 24bp, Tm is between 60 and 65, In every reaction, I perform the gradient melting temperature PCR. I also used hot-start Taq DNA polymerase, try to add DMSO to my PCR reaction mix in a 5% concentration. For every pair of primers, I try my best to avoid the same sequence between each other. I am very confusing, don't know should I design new primer again or continue to modify my PCR program.
Has anyone faced this problem? or there are something important I neglected? Thanks for your reply!

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Hi,

have you tried with different Mg2+ concentrations?

Cheers

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Thank you!
I have tried lots of Mg2+ concentrations, high to low. My be the melting temperature is too low, I think, I want to design new primers with Tm higher than 70c

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Have you tried running your PCR at different annealing temps?
Have you tried a different batch of dNTP?

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