How to design PCR primers in ChIP assay - (Jun/17/2006 )

hello everyone

i have not been here for a long time.
recently i will do some experiment related chromatin immunoprecipitation.
and the designation of the primer is a important part in the experiment
is there anyone who can tell me some experience on your primer design

hope for your suggestion

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I alaways follow the guidelines on the Applied biosystems website!

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For ChIP PCR priemr design, first rules for regular genomic DNA PCR should be followed.

Besides that, ChIP PCR has something special:

1. Usually focus is on a gene's promoter region which is mostly GC rich
2. To achieve high resolution, PCR product size should not be too big

Here are some general rules for designing ChIP primers:

• Primers should have a size of 20-24mer
• Primers should have a Tm of 58-60C
• GC content of 40-70%
• Avoid runs of an identical nucleotide (such as 'CCCC'), especially 'G's
• Keep amplified fragments between 150-250 bp
• Do a In-Silico PCR search or blast search make sure they amplify unique genomic region
• First test your primers on genomic DNA.
• Design at least 2 pairs of primers for each region of interest in case that some do not work

Hope that helps.

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