Help for cloning

Ms A F Simpson lsrig at csv.warwick.ac.uk
Fri Jun 19 10:48:54 EST 1998


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



More information about the Methods mailing list