Tricine buffer for PCR

[Michael Cooley]

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?

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[Frances Hannan (Zoology)]

szco...@chip.ucdavis.edu (Michael Cooley) writes:

>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?

Check out Nucleic Acids Research, 20, 623 for a comparison of buffers
containing Tris vs Tricine.

--
Frances Hannan
BILMS, Zoology, Downing St, Cambridge, CB2 3EJ, UK
Phone (0223)336663, FAX (0223)461954
f...@mole.bio.cam.ac.uk

[ell...@ucs.indiana.edu]

Michael Cooley (szco...@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.

[Jim Owens]

In article <3coqkb$g...@usenet.ucs.indiana.edu> , ell...@ucs.indiana.edu
writes:
>50 mM 2-mercaptoethanol

Have you ever left out the mercaptoethanol?

It or another reducing agent are not used by PE-Cetus in any of their
buffer formulations. It may not be necessary.

Traditionally 2-mercaptoethanol has been included in buffers for nick
translation because it was thought necessary for activity of DNA
polymerase I. It is not. There is only one cysteine in the monomer so a
disulfide bridge was not necessary for the molecule to be active. But it
did not hurt either.

Good luck,

Jim Owens

[Jasper Saris]

Michael Cooley (szco...@chip.ucdavis.edu) wrote:
: I hear tell of a buffer for PCR based on Tricine. It's supposed to hold a

Hoi Michael,

We use TRIS.HCL at pH8.4, pH8.9 or pH10 on roomtemperature. Most PCR sets
will work in all three buffers. Often the pH10 variant performs better
for smeary PCR's. The idea behind this is a higher annealing
(and elongation?) specificity of the several "Taq" polymerases we use.
Normal is 50mM KCl, 10mM TRIS, 0.5-2.5mM MgCl.

I hope this is helpfull information,

Jasper Saris

[Peter Gegenheimer]

Thanks for the grins! "Tris-HCl, pH 10" is another one of those weird urban
myths. Since the pH of pure Tris base is about 9 ( at 1 or 2 M), it will be a
humorous task to reduce it to pH 10. Also, of course, Messrs. H & H will spin in
their graves at the thought that such a beast could serve as a buffer...
(Ask if you don't know Messrs. H & H. Just doing my 2nd job as a Biochem prof!)
PAG.
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