Posted 23 November 2009 - 09:53 AM
I'm having a bit of a problem trying to make a buffer I need to follow a protocol I'm trying out. The protocol calls for a buffer containing 20 mM Tris-acetate (pH 7.9), 50 mM potassium acetate, 5 mM Na2EDTA, 1 mM dithiothreitol (DTT), 200 uM S-adenosyl-L-methionine, and some protease inhibitor.
I'm having trouble making the this buffer. I have some Tris-acetate (Sigma T1258), but when I make try to make it up, it's at ~pH 6.4. I would normally think that acetate buffers should be pH'ed with acetic acid, but that won't work here. I'm hesitant to use NaOH to raise the pH...
On the other hand, when one makes 50X TAE buffer, one combines 242 g Tris base and 57.1 ml glacial acetic acid (thus ~2 M) and 100 mL of 0.5 M EDTA at pH 8.0 (thus, ~50 mM) in a liter , and my recollection is that this buffer, when diluted to 1X, is around pH 8.3. Perhaps I should be making my Tris-acetate buffer up this way (by combining Tris base and acetic acid) rather than using the Tris-acetate powder from Sigma? Googling anything with Tris-acetate is overwhelmed by TAE and restriction enzyme buffers, I can not find an "add this much acetic acid to this much Tris base to make a Tris-acetate buffer of this pH" table...
I've already decided to forgo the DTT and add it to the reaction from a 10 mM stock just before use, and the SAM and protease inhibitor will likewise be added just before use. So, I'm trying to make a base buffer (probably 10X) that at 1X will be 20 mM Tris-acetate (pH 7.9), 50 mM potassium acetate, and 5 mM Na2EDTA.
I don't know why I'm so brain-dead this morning -- anyone got any advice?
Posted 23 November 2009 - 09:56 AM
Posted 23 November 2009 - 10:05 AM
Posted 23 November 2009 - 10:38 AM
2.43 grams of Tris base
0.6 ml of glacial acetic acid
pH this to 7.9 with additional glacial acetic acid, and bring it to a final volume of 100 ml, and call this 10X Tris-acetate (pH 7.9)?
Posted 23 November 2009 - 11:38 AM
remember, your buffer contains potassium acetate and sodium edta as well as tris-acetate, so either way of preparation should be fine (but, be consistent).
me, i would adjust the pH of tris base with acetate.
Edited by mdfenko, 23 November 2009 - 11:39 AM.
genius does what it must
i do what i get paid to do