Subcloning oligos into expression vectors
K.Mulcahy at sheffield.ac.uk
Tue Jan 21 03:56:12 EST 1997
David H. Gorski wrote:
> Thanks for the tip. Oddly enough, though, the NEB catalog doesn't state
> that you need that much flanking sequence for Nhe I to work.
I'm not sure if I agree with you on this. The NEB catalogue states
that Nhe I digestion of a ds-oligo comprised of the Nhe I site and 3
flanking base pairs on either side results in only 10% of the oligos
becoming cut after 2 hours or 50% after 20 hours. No data is given for
oligos with longer flanking sequences, but I assume they would be cut
more efficiently. Thus, you would definitely require more flanking
sequence than what is stated in the NEB catalogue (just 3 bp) in order
to get efficient cutting by Nhe I.
>I'm going to try to digest with Nhe I and Bgl II for several hours >and see if that makes a difference.
I'm not sure how heat sensitive the Nhe I enzyme is. If it is
inactivated after a few hours at 37°C you may also need to add another
aliquot of Nhe I midway through the digestion.
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