Ligation problems

Patrick F.H. Lai pfhlai at LOOKSMART.COM
Wed Jul 29 03:24:13 EST 1998


Dear Ian,

Just a few thoughts:
1.  Have you done Control ligations to see if it's your T4 ligase 
acting up ?
2.  Is your ligase buffer fresh ?  ATP in the buffer can die easily.
3.  How about coughing up a few hundred bucks for a 'real' TA-cloning
vector ?

Hope this is useful info.....
Good Luck !


Pat.Lai
<PFHLai at looksmart.com>
Grad. Student, U of Toronto


---- Begin Original Message ----
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 express=
ion
vector.  We have sheared it, blunted, ligated BstXI adaptors, and ligat=
ed
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 proteina=
se
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 i=
t
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


---- End Original Message ----




Hope this is useful info.   :-)


Patrick F.H. Lai  < PFHLai at looksmart.com >
Graduate Student
University of Toronto
Toronto, Ontario, Canada





       
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