question on cloning

Tom tsteenstrup at hotmail.com
Thu Jan 18 09:13:17 EST 2001


You may already be doing this, but since you don't mention it, here's a
couple of suggestions:

- Gel purify your insert and vector after the second digest
- Is the buffer compatible with both enzymes? If not then you have to EtOH
precipitate between digests or adjust the salt concentration.

Tom



"Sripathi Ramadurai" <sripathi at home.com> wrote in message
news:3A63E37A.F47154CC at home.com...
> 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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