Hiranya S. Roychowdhury
hroychow at nmsu.edu
Fri Feb 11 14:57:57 EST 2000
At 07:18 PM 2/11/00 +0000, S Hopkirk wrote:
>Hi, just wondering if you can help me with a couple of questions about oligo
>cloning, as I too am trying to cloning using this method.
>My your method describes dissolving oligos to 100pmol/uL.
>My PCR product is ~2000bp and I estimate from gel electrophoresis of purified
>DNA that I have it in solution at a concentration of 6ng/uL. How do I convert
>this to 100pmol/uL?
The avg MW of a ntd is 330pmol/pg
the rest is elementary:
For single stranded NA:
Where N= no.of ntd in the oligo
>From this, you should be able to make a standard solution. If you cant...
>Also when you precipitate the cut vector in EtOH, do you follow the method in
>Manniatis, and if so do you usually get to see a pellet once you have done
>this? When I precipitated 1ug of cut vector, the pellet wasn't visible.
A microgram of plasmid is a lot and should be visible as a pellet.
>"Frank O. Fackelmayer" wrote:
>> 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
>> Hi Caroline,
>> I´ve done oligo ligations quite often, and for my lab this procedure works
>> * dissolve UNKINASED oligos to 100pmol/ul in TE
>> * mix 5ul of both oligos, heat to 95C for 5 min, then cool slowly
>> * in a separate tube, precipitate 1ug of your cut vector with EtOH, allow
>> pellet to air-dry (do NOT dephosphorylate!)
>> * dissolve pellet in annealed oligos
>> * add 1.2ul of ligase buffer and 1ul of ligase (1U), then let stand
>> overnight at RT
>> * in your particular case, I´d add 10units of SmaI into the ligation mix to
>> cut vectors that have religated without taking up an insert. (of course
>> this works only if your oligo ends to not reconstitute a Sma-site, i.e. are
>> not 5´ GGG.....). During the overnight reaction, you´ll get more and more
>> vectors that have taken up an insert.
>> If you calculate it, you´ll see that the excess of oligos over vector is in
>> the 500-1000 fold. This is ok. We routinely get more than 90% clones with
>> exactly one insert. Remember that, as always, cloning success also depends
>> on the quality of your competent cells. Don´t use cells with competence
>> less than 5x10e8 (electroporation is usually good) for all cloning work
>> except for the most simple sticky-end subclonings.
>> Hope this helps,
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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