DNA polymerase that produces blunt end and doesnt smear! - (Apr/03/2008 )
I would appreciate it if anyone can name a good quality DNA polymerase that produces blunt end during PCR and doesnt smear!!
I have been using Invitrogen's Platinum Pfx polymerase and it has been giving me astronomical smearing on my gel. I have tried to reduce the smears by reducing PCR cycles from 30 to 25 (smear still there but my product is gone!), tried playing with Mg concentration (lowest was 1.5 mM where the smear was still there but my product was gone), tried playing with annealing temperature (from 60-68 Celsius), have tried reducing template concentration and tried altering ratios of forward and reverse primers. Did i mention that this was the 3rd round PCR using semi-nested primers? The 2nd round PCR was using full nested primers.
Can anyone please help? I would like to solve the mystery of Platinum Pfx OR get a new DNA polymerase! Any suggestions?
Thanks a gazillion times!
1. My experience with smearing gel is when I amplify a crappy DNA or RNA.
Either purity is low or degraded.
So check that out.
2. Incompatibility with the PCR buffer.
Certain template with certain G-C% is not suitable be amplify in certain buffer.
So try to use other product. You may try Qiagen or ABI. That works very well in my lab.
I read your post and I am having the same exact problem with Platinum Pfx that you had. Were you able to optimize this enzyme or did you find a new one that worked? Thanks for your help in advance.