Subcloning oligos into expression vectors

Kevin Mulcahy 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.

Best wishes,

Kevin.



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