I have potential dimerisation in all of my primers ranging from a 3 bp overlap to 6 bps.... The PCR is not working and I have what I believe to be evidence of dimerisation on the gel, but I'm not sure if enough base pairs overlap to create a dimer.
if you have a diffuse small ( around 50-80 bases) band then you probably have primer dimer. Before you design new primers try a hot start either with a hot start enzyme or by bringing the reaction mix without enzyme to 80c then adding enzyme manually and immediately start the pcr cycle then the temperature does not go low enough for PD to form and it may work. I have never liked 3 0r more bases at the 3' ens of the primers being complementary but primer pairs often surprise you and work well but when you have pd they amplify very well and remove all of the primer so pd often goes with weak or failed pcr. Try also using less primer...they are in huge excess so half or quarter should not affect the reaction but may cut down the pd formation but hot start is best
if you have a diffuse small ( around 50-80 bases) band then you probably have primer dimer. Before you design new primers try a hot start either with a hot start enzyme or by bringing the reaction mix without enzyme to 80c then adding enzyme manually and immediately start the pcr cycle then the temperature does not go low enough for PD to form and it may work. I have never liked 3 0r more bases at the 3' ens of the primers being complementary but primer pairs often surprise you and work well but when you have pd they amplify very well and remove all of the primer so pd often goes with weak or failed pcr. Try also using less primer...they are in huge excess so half or quarter should not affect the reaction but may cut down the pd formation but hot start is best
Dear Cate
I agree with Paul
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