How to modify a DNA construct?

Martin Hughes mhughes at fhs.csu.McMaster.CA
Mon Feb 12 21:00:02 EST 1996


On 12 Feb 1996, Gary K. wrote:

> > af930 at FreeNet.Carleton.CA (Changcun Jia) wrote:
> > 
> > >I would like to insert a 30 bp nucleotide into a construct. Anybody has
> > >any suggestion on how to do this?
> 
> In article <311d7cdb.42062718 at news.std.com>, rrohan at world.std.com (Richard
> Rohan) wrote:
> > Depends on if there is a unique restriction site at this location.  If
> > so, then you could use and adapter oligonucleotide.  Otherwise you
> > could PCR with oligos containing the 30 bp that you want to insert.
> > Rich Rohan> >
> The PCR route is the fastest but still requires a suitable restriction
> site for subcloning back into the original construct for replacement. 

Not necessarily!  You could use either EIPCR or (to blow my own trumpet)
a modification of a method published in the Feb issue of Biotechniques
(Hughes & Andrews - sorry, hav't seen a copy myself yet, so I can't give
you the page numbers!). - this way, all you need is one of a combination
of adjacent nucleotides, preferably GC, close to the site you want to 
insert you're sequence.

Cheers

Martin 



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