Ligating large constructs

Bruce Roe broe at aardvark.ucs.uoknor.edu
Fri Sep 17 06:51:00 EST 1993


In article <275ei7$9ho at tali.hsc.colorado.edu>, kroll_d at defiance.hsc.colorado.edu (David J. Kroll) writes...
> 
>Sorry if this has been discussed previously but I'm a new netter.
> 
>We're currently trying to subclone a rather large cDNA (4 kb) into an
>even larger baculovirus transfer vector (11.7 kb).  The insert has
>HindIII ends and the vector's been linearized with HindIII, phosphatased
>with shrimp AP, and gel purified.  Hence, there's little likelihood the
>ligation rxn is contaminated with phosphatase.  We use BRL T4 DNA ligase
>(1-2 units) and their rxn buffer which contains PEG.  We also supplement
>this with 1 mM ATP.  We've attempted the ligation about six times to date
>using 100 ng of vector and 3- to 10-fold greater molar amounts of insert
>but get no colonies.  In fact, our recircularized vector control has
>never given more than three colonies.  My phosphatase rxns have never
>given this low of a background with smaller plasmids.
> 
>I'm beginning to wonder if our ligation isn't the problem but rather that
>we can't get this large construct to transform DH5a.  I know that people
>have put human mitochondrial DNA into various plasmids and this construct
>would be about 20 kb.  However, I'm unaware if the rate-limiting step is
>the ligation or the transformation, or if there are particular tricks one
>must use in manipulating these larger plasmids.
> 
>Any advice on successfully obtaining my clone would be greatly
>appreciated.  Please respond by e-mail and I'll post a summary of
>suggestions.  TIA.
> 
>David Kroll
>University of Colorado School of Pharmacy
>Denver, CO 80262
>E-mail:  kroll_d at defiance.hsc.colorado.edu

David,
        Have you tried the following controls:
1. ligate your insert into HindIII/AP treated pUC? as a control for the
        ligation reaction done in parallel with the same insert and
        baculovirus vector (HindIII/AP) treated.  The pUC vector should
        have no problem with handling 4kb.
2. ligating your insert into HindIII treated pUC and baculovirus
        vector, neither of which was treated with AP.  There really
        is no need for AP treatment because you are only looking for
        a few colonies with a single insert.  So what if the background
        is high.
3. try NOT doing the gel purification.  It introduces more problems than
        it may be worth, especially with large vectors such as baculovirus.
        Just heat kill the HindIII or better yet, phenol extract after
        digestion followed by ether or chloroform extraction and ethanol
	pptn.
4. Since you are using the BRL reaction buffer, has it been frozen and
        thawed several times?  If so, you might want to add freshly
        made rATP instead of depending on the rATP in the "old" buffer.
5. Although there has been alot of discussion regarding ligation temp/time,
        recently on this newsgroup, a 12-24 hour incubation in a beaker
        of water placed in a refrigerator seems to give more optimal ligation
        than doing the reaction for a couple of hours at room temperature.
        Especially if you are having problems.
Cheers........and hope you get it to work,
--bruce
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