Making buffers - (Jun/23/2006 )

This is a very simple question but I need an answer.

When making buffers, if you have a recipe that says for instance:

300mM sucrose
3mM CaCl2
1mM DTT
10mM Tris-HCl, pH 8.0
0.1% Triton X-100

Does this mean that you make a stock solution of Tris-HCl at pH 8.0 and then add to the buffer to make 10mM? Or does it mean that the final pH of the ENTIRE solution is to be pH 8.0.

Thanks for any help.

Calvin

you will use a stock solution at pH 8.0, typicallly 1M

It is the Tris that is at pH 8.0 only. Make this as a stock (500mM or 1M). It should hold enough buffering capacity for the rest of the components to remain around pH8.0.

The recipe clearly states you should use Tris-Cl pH 8.0.
You should make a Tris-Cl solution, adjust the pH to 8 and use that one to prepare your buffer

Thanks for the help. That was my impression but the postdocs in my lab told me to adjust the pH afterwards. I would have assumed the recipe would have said to do that if that were the case so I just wanted to make sure.

10 mM tris does not have a lot of buffering capacity. the cacl solution may not be neutral and may throw off the pH of the solution. it wouldn't hurt to check the pH and adjust, if necessary.