Ligating large constructs

neale at mbcf.stjude.org neale at mbcf.stjude.org
Mon Sep 20 12:52:39 EST 1993


In article <275ei7$9ho at tali.hsc.colorado.edu>, David J. Kroll <kroll_d at defiance.hsc.colorado.edu> 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


We had trouble ligating inserts into a rather large plasmid (21 kb), basically
getting very few colonies as you described. 

In desparation I tried using 5-10 mM spermidine in the ligation and this did
the trick! My reasoning was that I thought there was potential for secondary
structure (in my plasmid at least) near the cloning site, and that this would
prevent accessibility to the insert during ligation. We had used spermidine
routinely in restriction digestions of lambda mini-prep DNA for that reason
too, ie remove potential secondary structure to get more complete digestion.

I have recommended this tip to others, but have not received any feedback. IMHO
it's worth a try.

Geoff Neale
St Jude Research Hospital
Memphis TN 



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