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Question on cloning

Zhonglin Chai Zhonglin.Chai at baker.edu.au
Tue Jan 16 01:20:51 EST 2001


As you vector self ligates, it means that your vector could be either cut by only one enzyme or the two restriction sites are compatible. Providing that both enzymes are working ok, you may want to check if the two restriction sites are overlapping, or too close to each other. Double digestion followed by a dephosphorylation step and gel-purification may be helpful to reduce the self-ligation background.

Hope this of help.

zhonlgin chai, PhD
Baker Medical Research Institute, Australia

>>> Sripathi Ramadurai <sripathi at home.com> 01/16/01 05:01pm >>>
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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