Tricine buffer for PCR

ellenmq at ucs.indiana.edu
Thu Dec 15 02:18:35 EST 1994


Michael Cooley (szcooley at chip.ucdavis.edu) wrote:
: I hear tell of a buffer for PCR based on Tricine. It's supposed to hold a 
: higher pH at the elevated temperatures of the PCR. Has anyone tried this 
: and does anyone know the recipe for making it?

Yes, it works very well for the Taq polymerase I use.
Here's the recipe that I use.
-Ellen Quardokus

10 X PCR Buffer (Tricine Buffer)

10X Concentration	Stock Solutions
300 mM			1  M Tricine, pH =8.4
20 mM			1  M MgCl2
0.1%			0.1% Gelatin (Difco)
1%			Thesit (Sigma P-9641)
50 mM			2-mercaptoethanol

1 M Tricine, pH = 8.4 (MW=179.2)
17.92 g tricine, 
Dissolve in 80 ml deionized water, pH to 8.4 with 10N NaOH.
(pH starts at ~5.1) Bring volume to 100 ml, autoclave.

1% gelatin
1 gram Bacto-Difco gelatin in 100 ml deionized water; autoclave and
mix.

1 M MgCl2 (MW=203.3)
20.33 g MgCl2 x 6H2O  dissolved in 100 ml deionized water; autoclave.

For 10 X Tricine Buffer (with 2 mM MgCl2)
3 ml 1 M Tricine, pH 8,4
1 ml 1% gelatin
200 ul 1 M MgCl2
5.7 ml deionized water (autoclaved)
100 ul Thesit (Polyoxyethylene 9 lauryl ether)
 35 ul 2-mercaptoethanol (14.3 M  standard stock concentration)
Total volume 10 ml buffer

It is better to freeze away 1 ml aliquots and to add the 
2-mercaptoethanol just prior to use; 3.5 ul per 1 ml of 10X Buffer. 

The buffer may also be made with no MgCl2 in order to do a MgCl2  
optimization of your PCR reaction.



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