How to design PCR primers in ChIP assay - (Jun/17/2006 )
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
I alaways follow the guidelines on the Applied biosystems website!
For ChIP PCR priemr design, first rules for regular genomic DNA PCR should be followed.
Besides that, ChIP PCR has something special:
- Usually focus is on a gene's promoter region which is mostly GC rich
- To achieve high resolution, PCR product size should not be too big
- 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