I am new to Chip-seq. Currently I am working on a bacterial transcriptional regulator. We do have a few putative binding target genes for the regulator. So I wanted to use qPCR to evalutate the purified DNA after immunoprecipation. I have run the gel and found the sonicated DNA fragments were ranged from 100-500 bp. The issue is that I am having a hard time designing primers for the promoter region of the target gene. For example, I have a gene with only 90 bp intergenic region upstream. I could not find any good primer pairs in the region. I was wondering shall I search primers further upstream or downstream of the region? Is it OK that the amplifed region for the gene is close to but doe not include the promoter region? Thank you in advance for your help!
You might be OK with detecting enrichment slightly away from the binding site, but a better approach is to increase the size of your qRTPCR product to include the target region if, for example, you are having problems with GC rich areas and primer design.
Thank you for your suggestion, Luke Norton. I was able to design a pair of primers spanning the whole intergenic regions, with a >200 bp PCR product though. Also I lowered Tm values for the primers since I am working on a low GC organism. I do have another question: For qPCR, do you do a series of dilutions of immunoprecipated DNA or use a pre-determined DNA concentration?
You will have to test this out in your own lab. Before library prep, we don't usually dilute too much for the PCR.
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