Help for cloning

niels hellings hellings at luc.ac.be
Wed Jun 24 06:01:31 EST 1998


In article <6me196$ksb$1 at primrose.csv.warwick.ac.uk>,
lsrig at csv.warwick.ac.uk (Ms A F Simpson) wrote:

> In article <3587DC49.77963829 at sciborg.uwaterloo>,
>         l4wang <l4wang at sciborg.uwaterloo> writes:
> >Hi, all:
> >I am cloning a cDNA into His tag fusion protein vector (Invitrogen). No
> >matter how I try,   the target cDNA won't get into vector. The strategy
> >for the cloning is very straight forward, double digested (NotI/EcoRI)
> >the target cDNA and the vector as well,
> 
> Since you don't say exactly which vector, I couldn't check,
> but are you sure that the NotI/EcoRI sites in the vector
> don't overlap?  If they do, only one of the two enzymes
> will cut, and the other won't be able to recognise the site.
> 
> Your digested insert will ligate to the vector at one end,
> but naturally won't be able to ligate at the other, since
> all the vector will be cut by one enzyme only.
> 
> >Li
> 
> love
> 
> Anna
 
Hi there,

it's also possible that your cDNA doesn't cut properly because the
restriction sites are at the ends; A possible solution for this is first
clone the cDNA in a TA cloning vector that has compatible restriction
sites, cutting the fragment out of this vector is much easier than cutting
the cDNA itself.

Hope this helps!

niels

--------------------------------------------
Niels Hellings, PhD student
MS research Unit             Tel 0032 (0) 11/26.92.07
Dr. L. Willems-Institute     Fax 0032 (0) 11/26.92.09
University Campus            E-mail hellings at luc.ac.be
B-3590 Diepenbeek
Belgium
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