ligation/cloning problem

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Thu Apr 4 09:18:53 EST 2002


I think DK's offered some good suggestions.  I'll just add my 2 cents as well.

When I've done this sort of ligation, i.e. an few kb insert into a big 
plant transformation vector, I've generally found that 100 ng insert in a 
20 ul reaction works well.  Usually when I'm doing anything out of the 
norm, I like to just empirically try differernt ratios of vector:insert 
ranging from 1:1 to 1:10.

As for cloning the insert into something like pGEM, I think that's a good 
idea.  For one thing, since you've generated your insert by PCR, you might 
want to sequence it to make sure that you've not introduced mutations into 
it.  It might be a bit easier to sequence out of something like pGEM than 
out of your plant vector.

If you're not already doing so, you might also want to transform by 
electroporation.  Efficiency of chemical transformation supposedly falls 
off quite sharply with such big plasmids.  I realize this is probably not 
your problem, but if in the end you have to play a numbers game, this might 
be helpful.

Hope these suggestions help as well.

Mike

Mike Sullivan
US Dairy Forage Research Center
Madison, WI


At 04:51 AM 4/4/2002 +0000, you wrote:
>jeremykapteyn at hotmail.com 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
>.

.
.
.

> >
> >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.
>
>I can't tell what is wrong - on paper everything is right and you should be
>getting lots of colonies. Assuming your screen by PCR works fine
>(did you do a control for this?), I'd suspect two possibilities:
>1. insert is toxic in the background of your plasmid.
>2. AP step. Somehow, don't know why, 90% of cloning mysteries I've
>heard of had this step included.
>
>Assuming it's not #1, I'd try:
>1. at least 20:1  _molar_ ratio of insert to on-CIAP vector.
>2. If this fails, clone insert into cloning vector like pGEM and then
>cut it out from there. I've seen people who say this was THE only
>way they could put insert in the "right"  plasmid. Might be that
>exact sequence 5` matters when it is too short (?).
>
>DK


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