question on cloning

Hallaril hallaril at hotmail.com
Wed Jan 17 04:51:59 EST 2001


Sripathi Ramadurai <sripathi at home.com> wrote:
> Hi!

> 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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