In article <3A63E31F.486F0C65 at home.com>,
Sripathi Ramadurai <sripathi at home.com> wrote:
>>to insert ratio of 1:3. Everytime, the vector self ligates and I do not
>get any clones with the insert. This means that my Restriction Enzyme
>digests are not working well. I double digest the vector and insert.
>I check them on a gel after the first digest. Once I find that the
>Restriction digest worked, I go ahead with the second digest. Is there
>any way I can know if the 2nd digest worked? Is there anything else that
The simplest way to find out if your second digest worked is to ligate.
You've done this and found that it doesn't work.
Two things: (1) Dephosphorylate your vector. That way, even the
single-cut vector can't religate, and you may be able to find the minority
of insert-containing plasmids.
(2) If you haven't already tried it, reverse the order of
your cuts; if the second enzyme cuts poorly, you may be able to see it
this way, and purify the digested version only.
Oh, and triple-check your sequences, to be sure that the sites really are
there, and (NB) are not overlapping, or too close to each other.
Ian York (iayork at panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England