It's a buffer for protein extraction from FFPE tissue, but it's not actually important for the answer. I just want to understand what to do exactly
I want to prepare 100 mL of this buffer:
2% SDS
20 mM Tris-HCl (pH 8)
I took 0,24 g of Tris and put it into the beaker, I added 0,1 mL of HCl, added 2 g of SDS and filled with dH₂O until I reached 100 mL
Now, my doubts are:
- Should I at first prepare 20 mM Tris-HCl and only after that add it to the 2 g of SDS until I reach the desired volume?
- Some people told me that actually "2% SDS" means that I have to first prepare a solution with 2% SDS and distilled water, and only after add it to a 20 mM Tris-HCl solution. If it's so, how can I understand how much volume of Tris-HCl and 2% SDS should I put in my solution to reach 100 mL?
Thanks for your help
If you want the pH of the solution to be exactly 8, then you should adjust the pH to 8 with HCl, added dropwise, after dissolving the 0.24 g of Tris and the 2 g of SDS in enough water to bring the volume to nearly 100 ml, leaving room for the addition of HCl to lower the pH. The pH adjustment should be done by using a calibrated pH meter and while magnetically stirring the solution.
An alternative would be to make the Tris-HCl (pH 8) solution first, adjusting the pH with a pH meter as above, then add 2 g of SDS to 100 ml of Tris solution. The slight increase in volume from the SDS can be ignored.
Wear a particle filter mask when weighing out SDS powder.
Adam B Shapiro Thanks for the answer! I know that I should adjust the pH, but in the lab I found other working protocols (about other things) that just use theorical values for Tris-HCl. So I was wondering if I could do some attempts without adjusting it (using 0,1 mL HCl), since the pH range described in many reviews is 7-9. It doesn't have to be so precise. Moreover the pHmeter in the lab isn't so reliable right now. Could this work anyway?
Considering the wide range of acceptable pH values, and the fact that the pKa of Tris is in the center of the range, if your calculation of the amount of HCl is correct, then it should be OK.
You did not say what the concentration of the HCl solution was. Concentrated HCl is about 11 M, so 0.1 ml contains about 1.1 millimoles.
100 ml of 20 mM Tris contains 2 millimoles.
The pKa of Tris is 8.1.
Based on the Henderson-Hasselbach equation, that should result in a pH of about 8.
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