PCR problems (using gDNA as template)

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liwei037
liwei037's picture
PCR problems (using gDNA as template)

I tried to get two fragments 3.0 kb and 1.6 kb from PCR using genomic DNA as template. But I only got 1.6 kb, and in the same gel the 3.0 kb smear. I used DMSO and Betaine, and I am sure that genomic DNA, primers, dNTP, KOD, and ddH2O are new, clean and work well. Other factors like temperature, cycles, concentration of MgSO4 are OK according to the instructions from Invitrogen. I ned your help, thanks a lot!

KWaraska
KWaraska's picture
Did you try increasing the

Did you try increasing the extension time to 3 minutes?

R Bishop
R Bishop's picture
If increasing the extension

If increasing the extension time does not work, I've had luck with increasing my concentration of polymerase (Taq, in my case) and/or lowering the gDNA template concentration.

Good luck

Please be sure to let us know what works for you in the end so others can benefit.

Rb