pcr with NH4+

David Micklem drm21 at mole.bio.cam.ac.uk
Thu Jun 26 11:48:19 EST 1997


In article <moellere.15.00A2F12D at pz350.ipsp.uni-hohenheim.de>,
moellere at pz350.ipsp.uni-hohenheim.de (EVELYN MOELLER) wrote:

>Hello:
>
>I would like to know if anyone on the net has experience with including NH4 
>ions (additional to MgCl2) into the PCR buffer and knows which concentrations 
>are useful....?
>I used the Quiagen PCR buffer which contains NH4 ions but the 
>company is not willing to inform customers about the concentration used in 
>their buffer. I will try  different concentrations now, but may be that anyone 
>already has done it and can indicate me a useful concentration/range.
>
>This buffer improves some of my crucial  PCRs. But I want to make my own 
>buffer instead of buying the expensive Quiagen kit (including the polymerase 
>and the Q-solution which I do not need) all the time..... and I think a 
>scientist should know what he/she is doing instead of using "undeclared" 
>products and supporting this fatalkit  philosophy....
>Thanks!
>Evely Moeller, University Hohenheim, Inst. Plant Breeding, Germany
>moellere at uni-hohenheim.de

AFAIK, NH4 buffers have been in widespread use for quite a while.

I have recipes for 10x buffers of: 160mM (NH4)2SO4, 670mM Tris pH8.8 at 25,
0.1% Tween20, +MgCl2 to taste.

KlenTaq (Barnes et al, for use in long PCR) came with buffer PC2, which at
10x is:

500mM Tris pH9.1, 160mM (NH4)2SO4, 25mM MgCl2 and 1.5mg/ml BSA.

This buffer has worked really really well for me for both regular and
mid-length PCRs.

If you have a look at recent posts (eg with www.dejanews.com), you may find
mention of betaine as a new wonder-additive, and the references I think.
Does anyone know if this is whats in Qiagen's new buffer.....

David

-- 
D.R.Micklem,                                Time flies like an arrow... 
Wellcome/CRC Institute,            Fruit flies like a banana.       
Cambridge CB2 1QR, UK               Email:drm21 at mole.bio.cam.ac.uk
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