PCR low amplification
Posted 06 February 2005 - 09:28 PM
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
Posted 06 February 2005 - 09:36 PM
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.
Posted 07 February 2005 - 02:04 AM
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.