Ligation or transformation problem

ChenHA hzhen at freeuk.com
Tue Jun 27 16:36:44 EST 2006


On 27 Jun 2006 10:45:45 -0700, "DY" <bearofthecity at gmail.com> wrote:

> 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?


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.






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