Amplification Problem - PCR Failure... (Nov/24/2003 )
Hi, I have designed 20 nt-primers whose Tm are 59°C and 60°C, respectively and I am having deep trouble in getting the desired product whose expected size is supposed to be 1,6 kb. I have tried a Mg++ gradient from 1,5 to 2,0 ĩM, but I only get unspecific products lower than the right size. Likewise, setting PCR at different annealing temperatures ranging from 55°C to 60°C was not effective. However, my PCR reagents and DNA template are working fine (checked that...). Could you please let me know whether you face the same problem and eventually guide me towards the answer ?
How did you design the primers?, using a DNA program?
What is your template, genomic DNA, plasmidic DNA?. It is obvious that your primers are not specific.
Have you checked homo and heterodimer formation?
why donīt you try "hotstart" protocol and see if itīs more specific or you can try Taq Gold from Promega (itīs quite good this enzyme). Good luck but donīt give up. thatīs what weīre all here for!!!!
Why don't you try a touchdown PCR for increasing specificity? or even try to adjust Mg concentration(between 1 to 5 mM), expecially if you're working with genomic DNA and with a cheap polymerase?
Good luck..and DON'T GIVE UP!
Actual PCR annealing temperatures are frequently placed 5°C below the predicted Tm, so 55°C may not be low enough.
you have quite a large segment to amplify, so what are your cycling times? Make sure you give your product enough time to be formed, you may have to go as much as 2mins for extension phase.