question on cloning

Dr. Mary Sarcina m.sarcina at ucl.ac.uk
Thu Jan 18 06:24:19 EST 2001


Hi,
I check you vector. sometimes you need to use another vector to stop
expression of your protein.
It might be that you don't get any clones because your protein is toxic,
when expressed it, to your system.

all the best
Mary

Sripathi Ramadurai wrote:

> 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

--
Dr. Mary Sarcina
Photosynthesis Research Group
University College London
Department of Biology
Gower Street
London WC1E 6BT


Phone:  0044-20-7679-7927
Fax:    0044-20-7679-7096
Email:   m.sarcina at ucl.ac.uk
www.ucl.ac.uk/biology/prg.htm







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