oligo cloning

[Hiranya S. Roychowdhury]

At 02:25 PM 2/7/00 -0500, Caroline Szymeczek-Seay wrote:
>I'm cloning (or trying to clone) a double stranded blunt ended oligo (a
>47mer) into pGL3 enhancer vector from Promega.  Vector is cut with SmaI
>and CIPed.  Oligos are kinased.  
>
>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'm getting no positives. 
>This is a little hairy also because there's no blue/white screening for
>this vector.  
>
>
>caroline
>

Be carefulwith that enzyme.. the CIP. Include a control ligation using a
larger/regular insert with the vector to determine if the CIP has been
completely removed/inactivated.
Other than that, any ratio you use for the ligation is just fine. What you
should be watching out for is the concatenation of oligos (almost a given
for blunt oligos) during successful ligations.


Dr. Hiranya Sankar Roychowdhury
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu

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