R. John Lye
rjl6n at virginia.edu
Mon Feb 7 15:54:22 EST 2000
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
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
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
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
not a 10X buffer as it is labelled (4 ul in a 20 ul reaction volume)).
rjl6n at Virginia.edu
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