Restriction Enzyme Help: Sac II

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Sun May 31 15:05:03 EST 2009


Hi Celeste,

if your fragments won't link, then something must be wrong with them,
i.e. they do not follow your theory.

One reason might be the enzyme has not cut (properly). Some enzymes
only can bind (and thus cut) to DNA when there is some overhang. The
NEB catalogue provides a nice chart where this effect has been tested
for with several enzymes. For myself, as oligo are cheap, I generally
add 4 or 5 bases extra (usually A's or T's) after an RE site which is
introduced by primers to avoid any hassle because of this.

Sometimes enzymes simply won't cut for unknown reasons: The
recognition sequence seems to be a necessary criterion but there might
be other features like secondary structure which from time to time
turn engineering into science and RE-search. In that case you'll be
stuck. You might try to add some DMSO, Betain, heat to your digest, as
one would do with noncompliant PCRs.

To find out if your PCR products have been cut well is quite
difficult, as they are quite long. If there is another RE site nearby
allowing to cut off a small fragment off the end of your PCR product,
you could check if the size of this small fragment changes after SacII
treatment. Use PAGE or capillary electrophoresis for this. This might
also tell you if you get partial digestion (i.e. if incubation time /
amount of enzyme / DNA concentration should be increased). You also
might label the ends with radioactive phosphate or a labeled base (eg
biotinylated dATP and Taq) and check if the label goes off by adding
SacII. But in the end there is a big chance that you'll just know why
it doesn't work and it won't get you much closer to a solution.

In terms of economy, the best solution might be making new primers and
use another enzyme, as another possible problem with SacII is that the
overhang is only 2 bases which is generally considered to be difficult
for ligation. If you need to design sort of unique RE site, SfiI is a
nice tool.

If you want to try again with SacII, also make sure that you add PEG
to your ligation, ligate at low temperature (i.e. in the fridge oder
set a cycler to 4-10 degC cycles). Also run the ligation quite dilute
in order to disfavor cyclisation if you have 2 SacII sites per
molecule).

Good luck!

Wo


More information about the Methods mailing list