question on cloning

Sripathi Ramadurai sripathi at home.com
Tue Jan 16 01:03:04 EST 2001


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?

All help is greatly appreciated.

Thanks!

Padmini








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