Template DNA for PCR- Concrentration or Volume?? - (May/01/2013 )
My question whether template DNA concentration should take precidence over template volume??
Heres my scenario:
1 Sample Processing: I have genomic DNA fragmented to ~200-600bp at a concentration of 15ng/uL. I load 1ug of this DNA (input DNA) into MethylMiner which essentially just enriches methylated portions of the input DNA (the methylated-enriched DNA is eluted at the end of methylminer with 80uL of appropriate buffer)
2) PCR Plate Loading: So now I run real-time PCR on the original Input DNA and the corresponding sample of captured methylated DNA, then calculate the ratio of their CT's to get a relative % of methylation for my amplicon.
The manufacturers protocol in MethylMiner only desribes adding 1-2uL of eluted DNA for PCR, but I became concerned when I checked the concentration of methylated DNA (on a Nanodrop 1000), and found samples only averaged between 2.9-5.6 ng/uL!
***Am I safe just loading 2 uL of Input DNA and 2 uL of methylated DNA for each PCR reaction?? (please say yes!) Or do I have to individually adjust allllll the template volumes for methylated DNA so they have the same amount of DNA per reaction as my input DNA??
Reaction Conditions (just in case it matters)
I'm running 10uL reactions in triplicate consisting of 5uL Sybr Green master mix, 200mM forward primer, 200mM reverse primer, 2.6uL nuclease free water, and then 2 uL of template.
Any help would be greatly appreciated!!
As you are running qPCR you should standardize the input for both the enriched and input samples. Personally I would adjust the input down to the level of the enriched, but it shouldn't matter which way you do it.