Question about primer design targeting GC-rich region - (Jun/09/2008 )
Hi, I need to design a pair of primers targeting a GC-rich region (just before the start codon) in the promoter of my favorite gene for the following ChIP assay. I have no other choice but this region since I need to see if some Sp factors bind to some motifs in this region. But it seems to be very difficult to design primers, the GC ratio of each primer is above 90%, and several consecutive GCs in my primer such as GGGG or CCCC. So there's no single band observed after I ran PCR even I raised annealing temperature to 66oC. I am wondering if anyone had any experience on this before? What shall I do?
Thank you very much!
Use a short primer, perhaps as short as 15 bp. Add betaine to the PCR reaction, 2-8%.
Betaine definitely. You could also design nested primers outside the GC-rich region. This may improve the primer characteristics.