PCR low amplification - (Feb/06/2005 )
I use specific primers to amplify a pathogen in total DNA extrations of sugarcane (pathogen + plant genomic DNA). the pcr worked very well early
but now i get faint bands only. i use about 150-200 ng of template ie. (pathogen + plant genomic DNA) for pathogen DNA content would be low.
i see template genomic DNA at the end of pcr when there is no amplification. but not when i get some amplification. why is this?
The only change i made is that i have dissollved the DNA in TE early i used to dissolve it in H2O. The bands I get are sharp and clear only that its not of the concentration it should be, I also get a faint background smear. I have no idea how to improve this any suggetions pls?
thanks in advance
try it again dissolving the DNA in H20... if it's the TE that's the problem.
if the bands are still faint, even in H20, make up another dilution of the primers... sometimes they degrade when exposed often.
that's about all i can think of, hope it helps.
I agree with vetticus as well, I suspect it is TE that is your problem.
What volume of DNA are you adding to the PCR? The EDTA will sequester Mg ions which are required for efficient PCR.
If you can not desalt your DNA samples I would suggest adding more MgCl2 to the PCR to take into account the EDTA removing the Mg from the PCR.