Patrick F.H. Lai
pfhlai at LOOKSMART.COM
Wed Jul 29 03:24:13 EST 1998
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
Hope this is useful info.....
Good Luck !
<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=
won't be offended at simple suggestions ...
We have purified some genomic DNA and we want to put it into an express=
vector. We have sheared it, blunted, ligated BstXI adaptors, and ligat=
into complementary BstXI sites (Invitrogen vector, Invitrogen adaptors)=
Result--very inefficient ligation.
Just blunting and ligating into dephosphorylated blunted
We tried using very degenerate primers and PCRing a smear up (there was=
reason for this, too complex to explain now) and tried using TA cloning=
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=
K treatment protocol to eliminate residual polymerase--no help.
We use NEB's T4 ligase, standard conditions as they recommend. We can =
gels on the ligation and we do not see evidence of ligation there, so i=
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
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 >
University of Toronto
Toronto, Ontario, Canada
LookSmart =85 or keep looking.
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