Melting tempreature

[mimila]

Can we exactly estimate the melting tempreature of the amplified product in real time,or it can be only done experimentally? Which progra could I use for check the tempreature?

[relaxin]

The reason to run melting curve for CYBR green qPCR is to check for purity of amplicon. So you have to do it experimentally. There should be a built-in program for the qPCR machine. Call for technical help from the manufacturer of the machine, if you do not know how to use the program.

[mchlbrmn]

There are programs to predict the Tm, but as far as I know they cannot be relied upon.
Primer3 program, in NCBI's PrimerBlast, gives a prediction of it's products (among other programs).
I never actually compared the predicted Tm's of these programs with the actual qPCR dissociation. It might be interesting to look back over my data some time. Actually, since I don't know the salt composition of my qPCR mix, it's probably proprietary, this would not give me an answer anyway. Also, due to pipetting error my Tm's often vary a bit in any case.
An eletrophoresis gel of the correct % for the size DNA is a more reliable way to check a PCR product.
One other point is that with larger PCR products in particular, sometimes a second melt peak can be formed when a lower GC% area melts before the rest of the DNA strand. I've never seen a progam even try to predict this.

[mimila]

Thank you very much, it really helped. I am interested how pippette error can change Tm value? I thought that is specific for the product? Could you explain this? Thank you:)