oligo cloning

[R. John Lye]

Caroline Szymeczek-Seay wrote:
> Vector is cut with SmaI and CIPed.  Oligos are kinased.

Be careful of over-CIAPing - in my experience, too much enzyme and/or
too
long a reaction will damage the ends of the DNA.  Make sure that all the
CIAP has been removed, also.  I usually use a little SDS/proteinase-K
digestion followed by phenol/chloroform to be sure it is all gone.
 
> My question:  is there a rule of thumb for amount of oligos to add to
> vector?  I'm trying a 10-fold molar excess, a 5-fold molar excess and an
> equimolar amt of oligo relative to vector. 

I generally aim for ratios of around 9 or 10 to one and 3 to one.  Blunt
end
cloning with short oligos is not terribly efficient, though.

>I'm getting no positives.

Are you getting any colonies with your "no insert" control; that is, is
your background clean?

Another thought - what ligase buffer are you using?  For blunt ended
cloning,
I like the PEG containing buffer from Methods in Enzymology, Volume 152
p. 109 (although there is a typo here, the buffer is actually a 5X
buffer
not a 10X buffer as it is labelled (4 ul in a 20 ul reaction volume)).

Good luck,
John Lye
rjl6n at Virginia.edu