influence of MgCl2 in PCR
Posted 18 January 2005 - 03:35 AM
Is it possible that there is still amplification with 10mM MgCl2?
And what exactly is the MgCl2 titration?
Posted 18 January 2005 - 05:34 AM
amplification is possible at 10mM Mg2+, in my oppinion. titration means you should try various concentrations of Mg2+ (e.g. 1, 3, 5, 7, 9 mM) for the same pcr reaction, since the concentration of Mg2+ influences the productivity and fidelity of polymerases. no pcr-product at 1mM Mg2+ and only smear at 10mM Mg2+ doesn't mean this reaction isn't going to work at all.
Posted 18 January 2005 - 07:06 AM
It really helped.
I have another question when it comes to the Polymerase.
I have also been varying the concentration of Taq Polymerase and I noticed that the Ct values are not really effected by an increase of concentration.( 1.25Units-31.25 Units)
Why is that the case?
Posted 18 January 2005 - 07:23 AM
Posted 18 January 2005 - 04:11 PM
Posted 19 January 2005 - 04:43 PM
At Ct, there is more enzyme than template regardless of the number of Units you are adding. Amplification is exponential until the number of molecules of enzyme euals that of template.
After 25 cycles of amplification, ABI expects 1.8 e 12 molecules of product.
The thermodynamic driving force for normal PCR is the molar excess of reagents with respect to template. Consumption of reagents other than the primers is not an issue. Limiting amounts of Taq DNA polymerse generates more artifacts than limiting primer.
More than 4 Units/100 µL reaction is not recommended as it can result in increased non-specific amplification and reduced yield of the desired product due to the intrinsic 5’ > 3’ exonuclease activity of Taq.
(M. J. Longley et al. 1990 NAR 18:7317, A. D. Sardelli 1993 Amplifications 9:1 and D. A. Bell and D. M. DeMarini 1991 NAR 19:5079.)