Help for cloning

niels hellings hellings at
Wed Jun 24 06:01:31 EST 1998

In article <6me196$ksb$1 at>,
lsrig at (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 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
B-3590 Diepenbeek

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