Restriction Enzyme Help: Sac II
L Celeste
I have been trying to amplify two segments ( 9 kb and 11 kb). I was able to
amplify the right-zed fragment with a primer set containing Sac II. After
restriction enzyme digestion with Sac II, I tried to ligate these two
fragments together. But they fail to ligate but the positve control worked.
Any help would be much appreciated. Thank you!
Celeste
WS
suppose you have removed Taq with a PCR cleanup.
if you check NEB's information on Sac II (http://www.neb.com/nebecomm/
products/productR0157.asp), you'll find 2 possible explanations:
1. Certain SacII sites (e.g. one in the right arm of λ DNA) are
resistant to cleavage. The reason particular SacII sites in λ DNA and
ΦX174 DNA are cleaved at significantly lower rates than those found
with other substrates is unclear at present.
2. SacII needs to interact with two copies of its recognition
sequence to cleave. As a result, substrates with single SacII sites
are cleaved at a reduced rate.
To overcome this, you might consider to increase enzyme concentration,
DNA concentration, and digestion time.
Have you enough overhang for SacII "to bite"?
Good luck!
Wo
L Celeste
Thank you for your info. How do I know if my DNA has SacII sites resistant
to cleavage? Also, I am not sure what you meant by 'Have you enough overhang
for SacII "to bite"?' Would you mind explaining that?
Thanks!
Celeste
2009/5/30 WS <novalid...@nurfuerspam.de>
> Hi Celeste,
>
> suppose you have removed Taq with a PCR cleanup.
>
> if you check NEB's information on Sac II (http://www.neb.com/nebecomm/
> products/productR0157.asp<http://www.neb.com/nebecomm/%0Aproducts/productR0157.asp>),
> you'll find 2 possible explanations:
>
> 1. Certain SacII sites (e.g. one in the right arm of λ DNA) are
> resistant to cleavage. The reason particular SacII sites in λ DNA and
> ΦX174 DNA are cleaved at significantly lower rates than those found
> with other substrates is unclear at present.
> 2. SacII needs to interact with two copies of its recognition
> sequence to cleave. As a result, substrates with single SacII sites
> are cleaved at a reduced rate.
>
> To overcome this, you might consider to increase enzyme concentration,
> DNA concentration, and digestion time.
>
> Have you enough overhang for SacII "to bite"?
>
> Good luck!
>
> Wo
>
> On May 30, 1:05 am, L Celeste <celest...@gmail.com> wrote:
> > Hi everyone,
> >
> > I have been trying to amplify two segments ( 9 kb and 11 kb). I was able
> to
> > amplify the right-zed fragment with a primer set containing Sac II. After
> > restriction enzyme digestion with Sac II, I tried to ligate these two
> > fragments together. But they fail to ligate but the positve control
> worked.
> > Any help would be much appreciated. Thank you!
> >
> > Celeste
>
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>
WS
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