ligation/cloning problem

D.K. dk at no.email.thankstospam.net
Wed Apr 3 23:51:04 EST 2002


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
>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.

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




More information about the Methods mailing list