Ligation problems

Ian A. York iayork at panix.com
Tue Jul 28 13:18:57 EST 1998


We're having a terrible time getting what should be a straightforward
ligation to work.  I have a feeling we neglecting something obvious, so I
won't be offended at simple suggestions ...

We have purified some genomic DNA and we want to put it into an expression
vector.  We have sheared it, blunted, ligated BstXI adaptors, and ligated
into complementary BstXI sites (Invitrogen vector, Invitrogen adaptors).
Result--very inefficient ligation.

Just blunting and ligating into dephosphorylated blunted
vector--inefficient.

We tried using very degenerate primers and PCRing a smear up (there was a
reason for this, too complex to explain now) and tried using TA cloning to
put into the vector (home-made TA cloning system).  Inefficient.

Tried blunting the PCR product and ligating into dephosphorylated
vector--inefficient. Blunting and BstXI adaptors/BstXI-cut
vector--inefficient.  For the PCR products, we tried using the proteinase
K treatment protocol to eliminate residual polymerase--no help. 

We use NEB's T4 ligase, standard conditions as they recommend.  We can run
gels on the ligation and we do not see evidence of ligation there, so it
probably isn't a question of inefficient transformation.  We've used
appropriate competent bugs (XL-1 from Statagene); test transformations
give acceptable results.
 
When I say "inefficient", I mean that we are orders of magnitude below
what we should be seeing.  

I tried this last fall and had no luck, set it aside, and a rotation
student is working on it now.  I've done several thousand ligations
without much trouble, and he should have beginner's luck, but it hasn't
made any difference.

As I say, I can't help thinking we're missing something obvious.  Any
suggestions welcome.

Ian 
-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England



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