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-   -   Ligation or transformation problem (http://www.molecularstation.com/forum/protocols-methods-forum/18601-ligation-transformation-problem.html)

Jayakumar, R 06-27-2006 07:20 PM

Ligation or transformation problem
 
Try adding CIAP into the restriction step with the vector (not INSERT).
This should eliminate any vector-vector ligation. But remember to
phenol:chloroform wash the vector and then elute it from DNA gel before
ligating with insert.
Jai


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[mailto:[Only registered and activated users can see links. Click Here To Register...].indiana.edu] On Behalf Of DY
Sent: Tuesday, June 27, 2006 1:46 PM
To: [Only registered and activated users can see links. Click Here To Register...]
Subject: 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?

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