ligation/cloning problem

Iva Toudjarska toudjarska at wi.mit.edu
Thu Apr 4 10:54:43 EST 2002


Hi Jeremy,

sometimes AP is damaging the ends of the vector. To make sure this is not the
problem you can just put one more control ligation reaction: Vector that has
been dephosphorilated, purified as usual + Ligase + Kinase = should give a lots
of colonies, if it doesn't make a new cleaner vector prep.
Just a reminder a full set of controls :
1. Cells + vector = transformation effitiency
2. Cells + Lig react of prepared vector without insert = will give you an idea
for the amount of undigested vector
3. Cells + Lig react of prepared vector without insert with Ligase = will give
you an idea for the amount of non dephosphorilated vector
4. Cells + Lig react of prepared vector without insert with Ligase and Kinase =
will give you an idea for any damage during AP step
5,6,7.... Cells + Lig react of prepared vector with Ligase and insert = your
ration experiment

good luck
Iva

jeremy wrote:

> 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
>
> <http://www.biowww.net/forum/read.php?f=1&i=5375&t=5375>
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