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PCR buffer with Tris-HCl and Ammonium sulfate (4:1)-does it exist?

PCR buffer with Tris-HCl and Ammonium sulfate (4:1)-does it exist? - Protocols and Methods Forum

PCR buffer with Tris-HCl and Ammonium sulfate (4:1)-does it exist? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 02-05-2004, 01:04 PM
Elisabet W
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Default PCR buffer with Tris-HCl and Ammonium sulfate (4:1)-does it exist?



Why is it Ammonium sulfate in my nested PCR mix?

Can you by a PCR buffer which contains Ammonium sulfate together with
Tris-HCL (pH 8.5), when the consentration is 4:1?

Or do I have to make these reagents by myself?

I use primers that are degenerated and contains deoxyionisine, if that
can help.

Elisabet W
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  #2  
Old 02-05-2004, 06:21 PM
Duncan Clark
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Default PCR buffer with Tris-HCl and Ammonium sulfate (4:1)-does it exist?

Historians believe that in newspost
<[Only registered users see links. ] > on Thu, 5 Feb 2004,
Elisabet W <[Only registered users see links. ]> penned the following literary
masterpiece:



I would not advise using a PCR buffer with more than 20mM AmmSO4 in the
1x mix so that would give a Tris of only 5mM. IMHO, that is
insufficient buffering capacity and I would not run below 10mM Tris.

I have seen and used a variety of buffers from 10mM Tris + 20mM AmmSO4
up to 75mM Tris + 20mM AmmSO4. pH from 8.3 to 9.0


Straight forward. Trizma preset from Sigma plus AmmSO4. Autoclave at no
more than a 10x stock with or without Mg. If using high Tris plus
AmmSO4, beware of pptation over time in the buffer. It will re-dissolve
when heated but can be disconcerting when one's previously clear buffer
turns cloudy.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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  #3  
Old 02-05-2004, 06:43 PM
Tom Anderson
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Default PCR buffer with Tris-HCl and Ammonium sulfate (4:1)-does itexist?

On 5 Feb 2004, Elisabet W wrote:


I believe the ammonium is thought to interact with the DNA and somehow
make the reaction less sensitive to annealing temperature, so you get more
product and less nonspecifics.


If you mean Tris:Ammonium 4:1, then i think Invitrogen do a buffer like
that: their PCR Optimizer kit is based on it. Fermentas do a 10X buffer
with 750 mM Tris and 200 mM ammonium sulphate, if that's any use; they say
they supply it with all their polymerases, so you might even have some
lying around.

tom

--
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL

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41does , ammonium , buffer , exist , pcr , sulfate , trishcl


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