smearing in PCR product - (Apr/26/2007 )
today I was trying to amplify 1.5kb fragment using genomic DNA as template and KOD DNA polymerase (Novagen). Quite strangly, all I got is bright smearing of DNA instead of distinct band. Can anybody help to explain why?
The KOD kit was newly opened and I set up the reaction on ice, following the protocol coming along with the kit. And I also called the technical support of Novagen. They suggested reducing times for annealing and extension steps, no final extenstion, no hot start, which I followed all. But still I got the smearing in the end.
I am just wondering if anybody who used this kit before had the same nightmare? any ideas to solve this?
thanks so much!
When I have smearing, I'll try the following first, I'll tweak the Mg conc in the PCR rxn. Also ensure that extension time is not too long (check the extension rate of your DNA polymerase). I'll prepare my rxn according to order... dH2O, buffer, Mg, dNTP, primers, template and DNA polymerase being added last. If it's not a Hot Start DNA polymerase, the rxn should be prepared on ice at all time.
Hopefully it'll work in your case.
Smearing could also be due to too much template or not so pure template
I thought smearing means degradation of any nucleic acid?
after tons of trials, I at last know why I got the smearing in PCR reaction--- the suggested concentration of Mg2+ in the buffer I was using was too high!!! I found that lowering the 3ul of 25mM Mg2+/50ul reaction mix to 1ul, I got the distinct band of desired size, although still with very slight smearing background.
And I am now thinking of raising the annealing temperature from the current lowest Tm-4C to Lowest Tm-2 or even to Tm, hopefully, that can help to reduce the background smearing.
so, thank you, "I love MSGs"--your tips hit the point!
I will keep you guys posted!