question on cloning
hallaril at hotmail.com
Wed Jan 17 04:51:59 EST 2001
Sripathi Ramadurai <sripathi at home.com> wrote:
> I am trying to clone a cDNA insert into an mammalian expression vector
> by directional cloning. I have tried ligating 3-4 times using a vector
> 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
> I am missing out?
Do it the otherway around.
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