ligation/cloning problem

jeremy jeremykapteyn at hotmail.com
Wed Apr 3 18:53:15 EST 2002


Hi,

I have been having trouble cloning a binary vector for plant transformation.

The plasmid vector is fairly large, ~17kb. I am attempting to insert a 2.5kb
cDNA non-directionally.

The plasmid is linearized with xbaI, after which I treat it with CIAP.
Ligations with CIAP vector vs non-AP treated vector subsequently run on an
agarose gel show a band of recircularized vector for the non-CIAP vector;
such a band is not apparent for the CIAP treated vector.

The cDNA insert was generated by PCR to engineer an xbaI site on one end of
the cDNA; the opposite end possessed one already just outside the cDNA
sequence. The xbaI site added by PCR was encoded in the 5' end of the primer
and there were 3bases from the 5' end to the cut site, so xbaI should not be
limited in efficiency due to too close proximity to 5' end (according to
Fermentas website). PCR product is xbaI digested.

Vector and insert are cleaned up using Promega Wizard kit and eluted with
water.

Ligations with insert alone subsequently run on gel yield what appears to be
insert monomers, dimers, trimers and tetramers, with faint band possibly
indicating circularized monomers and dimers also present. From this I
conclude that the insert has compatible xbaI ends.

The problem lies with successfully obtaining vector + insert ligation
products. I have attempted at 3:1 and 1:1 insert : vector ratios and with
various amounts of vector (100ng to 1ug per 10ul ligation).

The subsequent transformations and also dna gel analysis do not indicate any
positive results. The most recent transformation yielded low background
colonies with ligated CIAP vector without insert and the vector + insert
colony count was only ~30% higher. Seventy colonies screened by PCR produced
no positives, however.

I believe that the problem lies with the vector concentration and also the
molar ratio of vector:insert. I am hoping that someone can give me an
experience based suggestion so that I can avoid extensive empirical based
determination of the problem/solution.

JK


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