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PCR buffers - can I use MgCl2 alone (Feb/10/2006 )

I would like to use a minimum cation concentration in my PCR buffer solution. However there are so many possible combinations of different salts that can be used (K+, Na+, Mg++, KCl, MgCl2) . Is it possible to just use MgCl2 (and do I need to add Tris HCl).
Any help would be much appreciated!
Thanks!! blink.gif


you need either potassium or ammonium in there as well. Also you need Tris (~50mM) to buffer at pH8.5-9

-John Buckels-

QUOTE (John Buckels @ Feb 10 2006, 04:30 AM)
you need either potassium or ammonium in there as well. Also you need Tris (~50mM) to buffer at pH8.5-9

Thank you for your swift response!
So I could use an MgCl2 concentration of 1.5 - 50mM, KCl of 7-10mM and Tris-HCl of approximately 10mM (although the Tris-HCl conc can be altered depending on the pH)?
hmmmmm!! wink.gif


First generation 1x PCR buffers are essentially composed of 10 mM Tris-HCl, pH 8.3 and 50 mM KCl. Magnesium is added as required, but typically is 1.5 mM when the dNTPs are 200 uM each.

The Tris concentration can vary between 10 and 50 mM. While 10 mM is normal, the concentration cn be raised if the template pH is a problem. The pH is 8.3 at 20C so that at the extension temperature of 70C, the pH is

Each thermal DNA polymerase has its own optimum KCl concentration. The template length apparently has an effect on the KCl effect. Cheng et al. report that reducing potassium levels by 10-40% increased the efficiency of amplification of longer products. (Cheng et al. 1995 PCR Meth. Appl. 4:294) Like magnesium, potassium stabilizes primer annealing, including nonspecific annealing. Keep in mind that the enzyme's storage buffer usually contains 100 mM KCl.

Newer second generation PCR buffers combine KCl and (NH4)2SO4. Interactions between K+ and NH4+ allow primer hybridization over a broader range of temperatures. K+ binds to the phosphate groups on the DNA backbone and therefore stabilizes the annealing of the primers to the template. NH4+, which exists as both ammonium ion and ammonia under thermal cycling conditions, can interact with the hydrogen bonds between the bases to principally destabilize the weak hydrogen bonds of mismatched bases. The combined effect of the two cations maintains the high ratio of specific to nonspecific primer:template binding over a wide range of temperatures.

Magnesium concentation is a crucial factor in PCR amplification. Components in the reaction (template, chelating agents such as EDTA of citrate, dNTPs and proteins) affect the amount of free magnesium present in the reaction. DNA polymerases are inactive in the absence of magnesiou, while too much magnesium leads to higher levels of nonspecific amplification and decreased enzyme fidelity. The magnesium concentration is adjusted to give a constant free Mg++ concentration (of approximaately 1.2 µM) above the total dNTP concentration.

The role of Mg++ in PCR is dual: promoting DNA/DNA interactions and forming complexes with dNTPs that are the actual substrates for thermal DNA Polymerase. Typically, the dependence of the PCR yield on Mg++ is a bell curve with a broad maximum, but this can vary depending upon the particular enzyme. When Mg++ is too low, primers fail to anneal to the target DNA. When Mg++ is too high, the base pairing becomes too strong and the amplicon fails to denature completely when you heat to 94-99°C.

Lowering the magnesium concentration so that the enzyme barely processes will result in higher fidelity.


"salt free pcr buffers" are known LONG AGO 50mM Tris (pH 8.3) is used in Rapidcycler
visit and READ RAPIDCYCLIST smile.gif) you will find original citation for conventional cycler smile.gif)

" Second GENERATION " is IDEA to Mix Old Robost ammonium buffer ( tris + ammonium) with
bad KCl (Cetus) smile.gif)) Ammonium buffer was used since 1987-88 smile.gif)) and Always overperformed
Cetus because of Wide magnesium optimum smile.gif))

Another usefull site is smile.gif
Long PCR buffers MUST BE ROBOST smile.gif

Only do not even try to use this pHed stuff with
AmpliTaq Gold and various "chemical hotstart" Taqs smile.gif

Only do not even try to use this pHed stuff with
AmpliTaq Gold and various "chemical hotstart" Taqs smile.gif

Magnesium depends on the SPEED of Cycler smile.gif

1.5 mM was optimized for the First Generation of EXTREMELLY SLOW devises as PE 480
and various other 0.5 C / sec thermocy

New genereation of Heat block cyclers - SpeedCycler , ABI 9800 , Eppendorf work at speed 5-10 C/sec
and need more magnesium to SPEEDUP the reaction smile.gif)

How magnesium affect denaturation - visit Promega - find Tm Calculator- and Put different
concentrations of Magnesium- you will get Quantitative Data smile.gif in comparison
with changing KCl - only do not be very much surprized smile.gif

Super Idea to makeup 50mM KCl final using ONLY STORAGE BUFFER KCl smile.gif even 10 mM also super smile.gif even despite glycerol smile.gif