I am doing a Chip-seq experiment on one human sample and testing the DNA segment I got from Chip by qPCR first. What I found is my primer designed based on UCSC Genome Browser doesn't work very well for my sample, no amplification at all.
Then I tested my primers on genomic DNA, what I have found for the gene region, which should have around the same cq value since each of them are two copy are not very close to each other.
I am thinking this maybe the primers designed by the UCSC Genome Browser have some mismatch with my specific human sample. I wonder how should I improve my primers to make it works, and also what is the positive control for my primers?
Thanks!
Hi there, there are different ways you could design primers. I personally use either IDT technology [Tools, qPCR assay design, Primer-Quest], or NCBI Primer-BLAST, or sometimes even manually. Select the 400bp region where the peak is located and set to design primer pairs that will give the products between 100bp-150bp product. If the primers work, they should give the positive value already in input samples. Just make sure to run the qPCR product on an agarose gel. I am using UCSC genome browser (option BLAT) only to check that once I designed the primers, the product, specificity, and orientation is correct, helpful option. If they don’t work, just design another primer pair. I hope it helps! Good luck!
Hi, Jing Zhang .
I just come across the same problem with you do. I have a ChIP primer on my target gene promoter region (I've already blasted it in Ensembl), but when I use it for primer efficiency qpcr test by serial dilution input samples, it showed no amplification at all, even in the stock input DNA samples (the GAPDH positive primer have a avg ct value of 22 in stock input DNA samples).
Do you have any idea on this issue?
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