I have used primer set F1R1 for qPCR with syber green, in that I used 1ul Plasmid as positive control of published sequence and my cDNA as target. The Ct value for Plasmids was 18 and for cDNA was 20. The obtained Tm for Plasmid was 81 but for cDNA it was 76. The product finally ran on 3% gel, resulting in same sized band.
Then by sequencing the product we confirm that the sequence was different.
My question is that the melt curve difference depend only upon sequence difference or number of copied too, present in the product??
Dear Muhammad.. The Tm differences you observed result from sequence differences not copy number. It is possible to construct standard curves using known concentration of plasmid as a template fro real time PCR, in that case, every concentration will give exactly the same Tm... only the relative intensity will vary.
Tm = dH / [ dS + R ln(Keq) ]
dH (kcal/mol) and dS (cal/mol) are determined by sequence composition.
Keq is determined by concentration.
So technically yes, both can play a role in the observed Tm difference in the melt curve.
However, in practice, despite having different starting concentrations, at the end of the PCR, they will be at equivalent concentrations since you are using the same primers and same reaction conditions. So any observed Tm difference is largely from a difference in sequence composition (as was confirmed through sequencing).
These things are by no means absolute...and really should only be considered as indicative of how things are going unless conditions are judiciously controlled for.
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