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PCR works first time, then not! - (Aug/05/2004 )

Hi all,

I am testing a region that is near a highly repetitive region (not SINES or LINES) on human DNA. The PCR product size is only 138 bp. Everytime, the PCR works EXCELLENT the first time. Then soon (say in a couple of hours), it stops working! Everything is a smear (high MW, nothing around my band). Even my blank without any DNA shows the same pattern. It is not my reagents. Other primers are working good. And, it's not ME! "PCR king" of our lab also tried and failed! I checked the primer stocks on a gel and also measured OD. No problems there too! When I ran a PCR with no template and only one primer (eg. only forward primer), it gave me the same kind of smear. I checked the sequence for any secondary structure. A possible GG-CC looping can occur. So, I denatured that primer and did PCR again. There was reduction in the smear in the blank, but the template had the smear and not my band!

I have tried primers from Invitrogen as well as IDT. Did not make any difference. I make primers in water. As I am writing, a new reaction is going on where I have denatured the DNA before adding the PCR mix. If it does not work, I really do not know what else to try. Any one, any suggestions for this frustrated soul.

Thanks in advance.



Eureka! I solved the puzzle! Just did not check the complimentary values within and between the primer sequences. The two primers had high values!