PCR buffers for Taq and Pfu polymerase - (May/04/2006 )

Hello All,

I am using Taq DNA polymerase and Pfu DNA polymerase mix in 75:25 ratio. So far I used Taq polymerase buffer with KCl and without MgSO4 and then I would add MgCl2 seperately. Today I got stuck and got very little PCR product so dugg into it and I realised that there are tons of different Taq buffers offered. At the same time, there is totally different Pfu buffer. Following is the list is the list of buffers available.

1. 10X Taq Buffer with KCl
100mM Tris-HCl (pH 8.8 at 25°C),
500mM KCl,
0.8% Nonidet P40.

2. 10X Taq Buffer with KCl and 15mM MgCl2
100mM Tris-HCl (pH 8.8 at 25°C),
500mM KCl,
0.8% Nonidet P40,
15mM MgCl2.

3. 10X Taq Buffers with KCl Set
100mM Tris-HCl (pH 8.8 at 25°C),
500mM KCl,
0.8% Nonidet P40,
MgCl2 different in each buffer (12.5-30mM)

4. 10X Taq Buffer with (NH4)2SO4
750mM Tris-HCl (pH 8.8 at 25°C),
200mM (NH4)2SO4,
0.1% Tween 20.

5. 10X Taq Buffer with (NH4)2SO4 and 20mM MgCl2
750mM Tris-HCl (pH 8.8 at 25°C),
200mM (NH4)2SO4,
0.1% Tween 20,
20mM MgCl2.

6. 10X Taq Buffer without Detergent
100mM Tris-HCl (pH 8.8 at 25°C),
500mM KCl.

7. 10X Pfu Buffer with 20mM MgSO4
200mM Tris-HCl (pH 8.8 at 25°C), 100mM (NH4)2SO4, 100mM KCl, 1% (v/v) Triton X-100, 1mg/ml BSA and 20mM MgSO4.

8. 10X Pfu Buffer
200mM Tris-HCl (pH 8.8 at 25°C), 100mM (NH4)2SO4, 100mM KCl, 1% (v/v) Triton X-100, 1mg/ml BSA.

In these buffer systems, Tris-HCl concentration ranges from 100mM to 750mM and pretty much varying concentrations of the other components. Which one do you recommend to use for the 75:25 setup of Taq to Pfu DNA polymerase?

I know Pfu prefers sulphate over the others so its advisable to use MgSO4 rather than MgCl2 so I am not too sure if I should be using MgCl2 and (NH4)2SO4 mix so that there will be enough number os sulphates and Mg2+ in the mix!!!

This is driving me totally mad

In my experience, whenever there ar emultiple options, it doesn't really matter which one you choose. However, biochemistry isn't always like that, so why not try a few different buffers at the same time?
[Tris] doesn't really matter, justas long as your reaction is buffered. I'd be more worried about the Mg and SO4 problems. Try a couple of different Mg and SO4 buffer combos - one of them is bound to work.

I would use Buffer Number Five supplemented with 2 mM Magnesium Sulfate, final concentration. You might want to try 1 mg/ml BSA as well if you aren't getting enough product (BSA could cause smearing, however).

Triton X-100, Tween-20, NP-40 etc. are all relatively similar non-ionic detergents. Tris at 75 or 10 mM really doesn't matter, imho. 10 mM for buffering is way enough for PCR.

-Matt

Thanks guys for the replies.

I am more worried about Mg and SO4 but at the same time as Matt suggests buffer 5 doesn't contain KCl so would you care about that?
Ideally 500mM of KCl is required right?

The thing is, I know Vent works well in a buffer of similar constitution and I doubt the requirements for Pfu are that different from Vent.

In the future, I would look into buying Vent instead of mixing Pfu/Taq. Vent is probably cheaper.

-Matt

Hi I have experience using these enzymes and i think you shud go in for the Pfu buffer. If u can prepare please use PC2 buffer.

20mMTris HCl pH8.55 at 25 degrees C
BSA 150 mcg/ml
Ammonium sulphate 16mM
MgCl2 3.5mM
each dNTP250 micromolar

Hey pal,
also forgot to ask how big is this pdt you're tryin to amplify?