Question on cloning
sripathi at home.com
Tue Jan 16 01:01:34 EST 2001
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?
All help is greatly appreciated.
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