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