question on cloning
tsteenstrup at hotmail.com
Thu Jan 18 09:18:56 EST 2001
...and make sure to de-phosphorylate the vector! Shrimp Alkaline Phosphatase
works well and can be done simultaneously with the digest reaction.
"Tom" <tsteenstrup at hotmail.com> wrote in message
news:4SC96.102$Ab1.4130 at news.get2net.dk...
> 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.
> "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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