pHing Tris - (Apr/19/2006 )
I am setting out to make the tris solutions for the stacking and resolving gels (pH6.8 and 8.8 respectively (both solutions at 1.5M concentration) for westerns. What is the best way to go about making and pHing the solutions (I have heard that Tris buffers sometimes 'fool' cheaper electrodes and give the wrong pH)
Should I just make a 1.5M solution and by adding HCL/NaOH then pH using a meter and check with pH paper (can you get decimal point accuracy with pH paper?/is decimal point accuracy necessary?)
should I use TRIS-BASE powder or TRIS-HCL?
I was thinking of using the hendelson-Hakelbach equation but am not sure how to end up with a solution of the required pH AND molar concentration.
Any help would be greatly appreciated!!
you can't use a pH electrode that has a ag/agcl reference electrode and saturated agcl in the fill solution. an electrode with hg/hgcl reference and kcl (saturated or slightly lower, see instructions for electrode) or a solid state electrode without ag/agcl reference may be used.
the decimal in the pH is indeed very important.
you can start with either tris base or tris-hcl and adjust with hcl or naoh, respectively, or you can mix tris base and tris-hcl (of equal molarity) until you reach your desired pH (within +/- 1 pH unit from the pK of tris (8.2 or 8.3, depending on where you look)). this way your tris concentration will not change regardless of final volume.
thanks a lot!