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Newbie Questions - Protocols and Methods Forum

Newbie Questions - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 06-06-2005, 03:55 PM
p_u_c_k_o@yahoo.com
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Hey ppl, got some newbie question (roll eyes if u gotta):
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. Does
Tris-HCl play any part in the acidity of the buffer?
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?

These are probably really dumb questions, I don't work in a lab or do
double-digests in real life, just doing a microbiology question sheet.

Thanks

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  #2  
Old 06-06-2005, 10:13 PM
Don Incognito
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Default Newbie Questions

[Only registered users see links. ] wrote:

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


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


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

Don Incognito
Insert "spam" in the subject to reply by e-mail.

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  #3  
Old 06-10-2005, 09:59 PM
Scooter the Mighty
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Default Newbie Questions

<Hey ppl, got some newbie question (roll eyes if u gotta):
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.>

Enzyme manufacturers usually will give you a sheet telling you what
buffer to use for a double digest.

Personally, if it didn't look like I could do the digest with two
enzymes at the same time, I'd do one, precipitate the DNA, and do the
other in it's own buffer. Adding acid is difficult, both because it's
hard to pH 20 uL (or whatever), and because you'll be changing the salt
concentration.

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

Tris-HCL is the buffering part of the buffer, so yeah.

<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? >

I'd think that Na and K were close enough chemically that they would
usually be interchangeable, but there are no guarantees in life so you
would have to try it with whatever enzyme you're using.


These are probably really dumb questions, I don't work in a lab or do
double-digests in real life, just doing a microbiology question sheet.


Thanks

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