using PCR product as standard template - (Aug/16/2011 )
I want to use purified PCR product as my standard template in order to perform absolute quantification
after purification I quantified the purified product on the nanodrop and after calculating the concentraion of pcr product by nanodrop i calcilated the copy number per ul of my samples by this formula:
molecules of DNA= Mass(in grams)* Avogadro's number/Avrages mol.wt.of a base * template length
then I prepared 10fold serial dilutions of my purified product and used this as my standard for absolute qPCR
but the result is not good.
most of the time i have no band on the gel for seial dilution
first i set the qpcr with genomic DNA after getting good melt curve and one specifice Tm i repeated this condition for both genomic DNS(unknwon sample) and standards but the Tm of stundards are different from the sample ' tm
so i have some problems: 1 diffrence in Tm beween sample and standards
2 no band on gel for standards
3 high efficiency (i used 10 fold dilution 10^7-10^3 copy number per ul)
4 when i chenge the pcr condition to get the specifice tm efficincy chenges too
i guess pcr product are sensitive to temprature (denaturation time affects the melte curve for standards) and i guess to there is some inhibitors that prevent of the progress of pcr of standards
can anybody help me ?? and give me some guidance with problems?
PCR amplification is highly dependent on the annealing temperature of the primers to your template. Therefore, it is possible that while the temperature you used for the amplification of the genomic sample was good it wasn't in the good range for your standard template leading to uncomparable yields to your original standard template amplification. Thus, making it not a good standard for absolute quantification of this sample. A possible way around that problem is to design primers with roughly the same annealing temperatures for both standard and sample.
I would not re-amplify a pcr product with the same primers. You could use internal primers on a diluted pcr sample, but pcr products, even if purified, have many short products that can confuse your quantitation.
Use of PCR products as standards is not usually recommended for a longer storage. They can be used, but only really fresh, because they degrade on the ends in time. Other thing is that standards containing only the desired sequence amplify with different efficiency than gDNA, because in gDNA you have lot's of "dummy" DNA present, that is not amplified. Diluting the standards in buffer containing 20ng/ul rRNA or other dummy fill-in can mimic this effect. I once used standards like that, but I cut the specific product from the gel, which should eliminate the presence of other than specified sequence.
A wild guess.. I was thinking some time ago about the Mg2+ concentration that can be relatively changed in the presence or absence of nucleic acid, which traps the Mg ions. In your DNA sample you have a DNA concentration, the diluted standard has many copies but little actual mass, so more free Mg2+. Could the different Mg2+ concentration change the Tm of your standards? I don't know, but I find this idea interesting.
Also you probably have too high efficiency due to the inhibition in the more concentrated standards, that skewes the standard curve.