Newbie Questions

[Don Incognito]

p_u_c_k_o at yahoo.com wrote:
> For a double digest, when the optimal pHs of the two restriction
> enzymes are different, do you do the higher pH (less acidic) reaction
> first, then add a little acid to meet the second optimal pH.

In "real life", you would find a buffer that would work acceptably for
both. The specifics, of course, depend on the endonucleases in
question.

> Does Tris-HCl play any part in the acidity of the buffer?

Possibly (depending on the pHs and quantities of the respective
buffers).

> Also, I've heard you can exchange NaCl with a mixture of NaCl/KCl (as
> long as the total concentrations remain the same), can you replace the
> NaCl completely with KCl?

Not unless you're sure that K+ ions won't, say, inhibit the
endonuclease or increase its star activity (promiscuity of cleavage).

Don Incognito
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