Double digest Sma1/Sac1
hroychow at NMSU.EDU
Mon Jul 6 14:55:35 EST 1998
At 11:28 AM 7/6/98 -0700, Bernard Murray wrote:
>In article <kirkpat-0607981203180001 at gietz.hgen.umanitoba.ca>,
>kirkpat@?cc.umanitoba.ca? (Rob Kirkpatrick) wrote:
>> In article <6nqn62$pb3 at is.bbsrc.ac.uk>, "Andy Bettany"
>> <andrew.bettany at bbsrc.ac.uk> wrote:
>> > Dear All,
>> > I need to double digest a plasmid with Sac1 and Sma1.<snip>...
>Okay, just to confuse matters I'll venture another opinion.
>I'd suggest digesting first with SacI alone at 37degC and
>then dropping the temperature and adding the SmaI.
>My reason is that SmaI can nibble back at warmer temperatures
>and you may not finish up with the exact ends that you
>intended. If you look back through the archives you'll
>find a few posts on digests with SmaI (I believe some people
>even suggest dropping the temperature to ambient or below).
> I hope one of the methods gives you what you want,
Now, I have occassionally come across postings re: SmaI digests and all the
"horror" stories about it. I use SmaI-digested vector and inserts almost
routinely for sequencing of subclones. I have carried out SmaI digestion in
all kinds of buffers that come with all kinds of SmaI preps (you know, BRL,
NEB, Pharmacia etc.) at 37C (routinely) and have never observed any "chew
back" by SmaI! I have been monitoring the sequences at the junctions closely
for a couple of years now since I first came across such comment(s) about
the enzyme. I can emphatically say that SmaI has never done that in my hands.
As far as double digestion goes, I am quite comfortable digesting
with two enzymes simultaneously in a compatible buffer after making sure
that the sites are well separated (at least 6bases). The "One-Phor-All plus"
buffer (pharmacia's) is a very good one that works with almost all common
enzymes with slight variations for those enzymes that require lower or
higher salts (eg. using 0.5x to 2x).
Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu
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