Hi Wo,
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 <novalidaddress from 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... 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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