adjusting pH of 4x stacking buffer - (Aug/14/2012 )
I am following a protocol that requires the stacking buffer be adjusted to a pH of 6.8. I accidentally overadjusted it to around 6.1 so I used some NaOH to compensate the pH back to 6.8. Would this affect the stacking gel performance or not really?
The NaOH adds Na to your solution (the OH- combines with the H+ in the solution to form water), if the assay is dependent on salt you may have a problem, but it should be fine most of the time.
The usual way to do this sort of thing is to make a tris solution of pH 6.8 and add that to your stacking buffer.
the addition of naoh will affect the stacking buffer.
if you overshoot then you should add more basic component (tris), calculated for a greater volume, readjust pH, then bring to the new final volume.
Ack! OK thanks a lot for replying - I haven't used it yet but now I will dump it out and remake it and be careful about adjusting pH.