Melting Temp of Standards Changed? - (Jul/07/2011 )
I am trying to use a standard curve method for qPCR quantification. To do this, I generated PCR products using the same primers that I will use for qPCR using AB's Fast SYBR Green Master Mix and StepOnePlus machine. Then I purified the products using Qiagen's Qiaquick PCR Purification Kit and measured DNA content with nanodrop before making my dilution series of "Standards".
The trouble is that when I generated the standards, the melting temperature for the product was about 85C, however when I use these standards for qPCR the new melting temperature is about 74C, while the melting temperature for my samples is 85C.
Is there an easy explanation for this?
Tm is highly dependent on salt conditions. Are your purified samples dissolved in the same buffers as your test samples?
phage434 on Thu Jul 7 18:46:12 2011 said:
Tm is highly dependent on salt conditions. Are your purified samples dissolved in the same buffers as your test samples?
Both are in TE, which gets dilluted 1:2 in water and then 1:2 again in Master Mix. I am guessing that my standards are too dilute, so there are only primer dimers being formed. I think I need to run some agarose gels to look at the sizes of products etc.
Quick follow-up: Whenever I generate PCR products using Fast SYBR Green Master Mix and then run them out on a gel, they appear as diffuse and "fuzzy" smears. The same PCRs run with NEB OneTaq Master Mix are clean single bands. The qPCR data look nice, but I just don't understand why I am not getting clean PCR products.