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pcr 10X buffer preparation


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#1 vincio

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Posted 28 April 2013 - 03:42 AM

Hello,
I am tryng to prepare home made 10x buffer (750mM tris ph 9, 500mM KCL and 200mM NH4SO4, 1% tritonX). My peparation is not working and I have two doubts to resolve:

1) I am using Tris-HCL solution 2M ph 9 made from tris-hcl salt bring to ph with NaOH : is it this salt correct for PCR? Have I to use Tris-base ? I am thinking in some negative iteration of Na+ ions..

2)I use ph 9 tris but in final solution the ph is 8.0-8.2 I have to further adjust ph ?

thank you for any help

Vincenzo

#2 bob1

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Posted 28 April 2013 - 04:02 PM

Yes, you need to use the tris base - the addition of NaOH will change the Na+ concentration, which could alter the folding of the enzyme.

Don't worry about the pH in the 1x solution, it is more critical in the 10x to ensure that the pH is correct in the 1x.

#3 vincio

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Posted 29 April 2013 - 09:47 PM

Yes, you need to use the tris base - the addition of NaOH will change the Na+ concentration, which could alter the folding of the enzyme.

Don't worry about the pH in the 1x solution, it is more critical in the 10x to ensure that the pH is correct in the 1x.

Thanks!




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