No PFU PCR Amplification! help! why?

[ale]

i am really having great problems doing an amplification with pfu and it is really important for me because it is important for the experiments for my university thesis. if you have ever worked with the pfu doing an amplification give me the suggestions so that i can try them because i am really going crazy for this thing.

[moleculardude]

Hello Ale!
have you tried the PCR with regular Taq polymerase or Hotstart?
Also how was your DNA template quality? Could it be contaminated / degraded?

What is the source of your DNA?

Cheers!

[̑̑̑]

i would only use pfu if i wanted fidelity... as md suggests try taq to start with (its cheaper) and once you have optimised you can try pfu... and check your template for possible degradation... also you could try touchdown pcr to find optimal conditions... i cant say that much with the details you are giving...

[Paulo Henrique]

Hi, I`m from Brazil, and I am having problems with pcr with regular taq. My teacher said that I had many DNA template in the reaction and wants to know why this is happening. Can somebody help-me?

[danfive]

Originally Posted by Paulo Henrique

Hi, I`m from Brazil, and I am having problems with pcr with regular taq. My teacher said that I had many DNA template in the reaction and wants to know why this is happening. Can somebody help-me?

To much DNA can inhibit the PCR reaction by binding all the MGCl2. So do a dilution series or dna quantitation. You only need a small amount of dna template. Good luck!

[Paulo Henrique]

Ok, I`ll Try. Thank you very much for the help