cloning oligos

Bernard Heymann bheymann at bragg.bio.purdue.edu
Tue Dec 27 10:52:04 EST 1994


In article
<Pine.SUN.3.91.941226135639.1870E-100000 at namaste.cc.columbia.edu>, Eric
Daniel Slosberg <eds9 at columbia.edu> wrote:

> 
> Hello:
> 	Does anyone have experience/knowledge about cloning ss 
> oligonucleotides into a vector.  Do I need to make a complementary oligo 
> and anneal to make a dslinker?  Is it possible to just ligate the oligo 
> into the vector and fill in the second strand w/klenow?  Also what 
> ratio of insert:vector is needed for this type of cloning (especially if 
> I make a blunt ended dslinker). Any advice can be posted on this newsgroup. 
> Thanks in advance.

I've done this kind of cassette cloning several times now, with great
success.
However, I haven't tried doing it from a single ss oligo, which seems to be
a long shot. I design the two complementary oligos with the appropriate
sticky ends, phosphorylate them with T4 kinase separately, anneal them, and
ligate them into the receiving plasmid like any fragment. The amount of
oligo I use is around 0.5 to 1 nmol. I usually design a restriction site
into the cassette, so that I can assay for its insertion. Otherwise, I can
assay for its insertion and orientation by PCR. I hope this helps. Good
luck.

-- 
Bernard Heymann
bheymann at bragg.bio.purdue.edu



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