PCR buffer and PCR efficiency - buffer pH and salt (Mar/14/2007 )
I am just wandering there are so many type of PCR buffer in the market.
Some using KCl, some using NH4SO4 and with different pH.
Can some one tell me what is the different between KCl buffer and NH4 buffer? Which one better?
How PCR buffer pH can affect PCR efficiency?
What is the optimum pH for PCR? 8.0, 8.5 or 9.0?
First generation 10x Taq buffers typically consist of: 0.1 mM Tris-HCl, pH 8.3 ± 0.05, 0.5 M KCl.
Second generation 10x buffers consist of approximately 0.2 mM Tris-HCl, pH 8.8 ± 0.05, 0.1 M KCl, 0.1 M (NH4)2SO4, 1% (v/v) Triton X-100.
MgCl2 is added as required.
Buffers with higher pH have been proposed to enhance long PCR reactions by facilitating template denaturation and reducing DNA damage (Barnes, Proc. Natl. Acad. Sci. 91:2216-20, 1994; Cheng et al., Proc. Natl. Acad. Sci. 91:5659, 1994).
Some recipes include BSA, which reduces the amount of template sticking to the side of the tube, making it available for amplification and reducing the risk of primer dimer.
The (NH4)2SO4 buffers are reported to permit consistent PCR product yield and specificity over a wide range of magnesium concentration (1.0 to 4.0 mM MgCl2). Also reported to improve overall specificity and yield of PCR product when compared to first generation PCR buffer. Interactions between K+ and NH4+ are reported to allow specific primer hybridization over a broader range of temperatures.
Thank you for your explanation.
here I have two question to ask:-
1. as you said higher pH enhance long PCR reaction....
how long consider as long? 2000bp? or 20kb?
what is the function of K+ and NH4+ in PCR buffer?