problem with my PCR - primer dimer. - (Apr/18/2005 )
I'm trying to do 3'RACE. (I have already got the 5' end using specific primers). following the RT-PCR using an olido dT, I did PCR with my Specific primer (17bp) and anchor. only the PCR reaction using the anchor primers alone showed a product, however using both primers did not show the product I need except a strong 80bp band (primer dimer??). My product size should be around 200-300bp.
I thought that my DNA copies are very low, or the primers form a dimer.
I have already tried: hot start PCR, touchdoun PCR, gradient PCR and PCR using 50 cycles, and specific different primers.
what else can I try???
thanx for any help!!!!!!!
It seems that your GSP didn't work well in your 3'RACE. Why not design another GSP and use the current GSP as the nested primer? so if you have a new GSP ( The expected 3'RACE Product should be longer than 300 bp---which is the expected size of your current GSP with 3' Universal primer), you could use the PCR product as the template and amplify again with the nested primer ( to be exactly, the current GSP and 3'UP). It's normal that you got a few unspecific product from RACE. so it's important to use the nested primer to increase the specificity. good luck.