Ligation or transformation problem

I just have a very puzzling problem when trying to clone a 700 bp gene
into a vector. The vector is about 5.8kb. I PCRed the gene, which works
beautifully. Then I digested both the insert and vector using BamHI and
XhoI. The DNA was gel purified. Everything seems OK.
However, after I ran the ligation and used the ligation mixture to
transform E. coli cells, I did not get a single colony!
I did my ligation using Roche rapid ligation kit. Since I did not get
colonies, I went back to repeat the ligation experiment and checked the
ligation mixture on a gel. I also did a control experiment with both
insert and vector, as well as the ligation buffer, but omit the ligase.
On the gel, I found the control mixture contains both the linear vector
and insert, as I expected. For the mixture with ligase, I saw a smeared
band with high molecular weight (higher than the linear vector) and
both the bands correponding to the linear vector and insert
disappeared. Therefore, I concluded that at least the ligase works and
both the insert and vector can be ligated into something, though I
cannot say they can be ligated into the product I want.
However, I still have no colonies, even use ligation mixture that I
clearly proved to contain some products with above method.
I also set a ligation reaction with only the vector. Normally, two
vectors should be ligated together and this should give you some
colonies (though these colonies are useless for cloning purpose).
However, I still have no colonies.
Now I start to suspect the competency of my cells. But these are
commercially availiable competent cells with good tracking record in
our lab. AND, I also did a positve control using intact vector during
the transformation, which leads to lots of colonies. Therefore, the
cells seem to be OK.

Right now, I do not have any more ideas on how it should work. Do you
have more ideas how to trouble shoot this problem?

>Right now, I do not have any more ideas on how it should work. Do you

I have never tried this method, however I found it quite interesting
if you'd like to try: an in-gel ligation technique....I am attaching
the "protocol".

pow



On 27 Jun 2006 10:45:45 -0700, DY <[Only registered users see links. ]> wrote:

On 27 Jun 2006 10:45:45 -0700, "DY" <[Only registered users see links. ]> wrote:



Possiblities -

1) You have a protein which is difficult to clone, perhaps due to
toxicity of the protein, or perhaps unusual sequences.

3) You use too high a DNA concentration for ligation - many people
assume that the more DNA you add, the more effective the ligation will
be. Not true for ligation. The plasmid need to circularise, at high
DNA concentration it will tend to form concatemers, the smear you see
is probably an indication of that. Do not use more than 100 ng DNA
per reaction.

3) Your DNA is not digested properly - add extra bases to the oligos
you designed to ensure that the DNA is digested properly. Check NEB
catalog for the required number of bases to add. BamHI requires 2
extra bases and XhoI I use at least 4. Also check your vector
sequence, sometimes the restriction sites are too close together, in
which case you digest the vector with enzyme that require more extra
bases first (XhoI in your case), then the fewer one.

4) For directional cloning (i.e. cloning into 2 restriction sites), DO
NOT use CIAP as someone else said. It can cause more problem than it
will solve and is completely unnecessary in any case for directional
cloning if you make sure that your vector is cut properly.

The key to any cloning experiment is reducing the number of steps prior
to the ligation, to avoid losses of DNA and sources of degradation. We
routinely do cloning of PCR fragments with the following scheme:
1) PCR amplify fragment so that only one band is visible (try to
optimize PCR by ordering good primers and different temps)
2) instead of gel purification, we purify the entire PCR mix using
Qiagen column kit
3) digest vector and PCR fragment
4) precipitate vector with ethanol
5) column purify PCR fragment
6) check everything on gel, estimate concentrations of DNA and vector,
use appropriate amount of DNA in ligation. vector DNA =50-400 ng, use
as much pcr product as possible (in vast excess) in a total of 20 ul.
We use ligase from Fermentas, high concentration type.
Ligate overnight, 16 degrees.
7) electroporate

Since you are getting no colonies I'm wondering if your ligase is dead.

Good luck