Restriction Enzyme Help: Sac II

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Sat May 30 05:12:54 EST 2009


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), 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... from 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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