cloning oligos

Ken Saville saville at umail.umd.edu
Wed Jan 4 14:50:10 EST 1995


.Eric Daniel Slosberg (eds9 at columbia.edu) wrote:
>       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?

>In my experience it is very difficult if not impossible to link a single-
>stranded oligo to a staggered vector end. (Maybe I'm just not able to do
>it.) Using two complementary oligos works like a dream.

>For more advice using linkers see one of the bibles, either Maniatis or
>Current Protocols.

>Hope that helps,
>-Cornelius.

eric,

I recently cloned 40 bp oligo into pUC19.  The oligo was designed so
that it overlapped the HinDIII site at the 5' end and the SacI site at
the 3' end.  The oligo was also 5' phosphorylated.  pUC19 was digested
with HindII and SacI.  I found that a large ecess of oligo (> 1000
molar excess) was needed for succesful ligation.  However, no
subsequent treatment was required.  That is, I just transformed E.coli
by electroporation, and let the bacteria fill in the gap.  It worked
the first time with no trouble shooting needed (the molar excess was
actually done by mistake).  having said that I have recently tried the
same thing with a 16-mer and it didn't work the first time.  I have
repeated it, again with a large excess of oligo, and have 3 putative
clones.  i will have the sequence Friday to confirm.  i'll let you know
if it works.

cheers, 
ken       




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