Cloning lab
The cloning lab is set up to fit into time constraints. It does not reflect the order of
experiments normally done to clone a foreign gene into a plasmid and get it
replicating in E. coli.
The normal order would be:
1. purify the vector DNA (pBluescript KS+II)
2. Purify the DNA that is the source of the insert (The CAT plasmid, called
pUC9CAT)
3. cut the vector DNA with BamH1 and HindIII enzyme. This will release a small
fragment with the sequences of the multiple cloning site and a large fragment
(3.0 kb). The large fragment will be almost the same size as the vector cut with
either BamH1 alone or HindIII alone (See the control gel in the Cloning gel
results powerpoint).
4. Separate the BamH1-HindIII cut vector from the small fragment by
electrophoresis through an agarose gel. It is nearly impossible to separate the
BamH1-HindIII cut plasmid from any contaminant that may be cut only at the
HindIII site or at the BamH1 site. These could potentially religate in the negative
control ligation you did in the lab class.
5. cut out the section of agarose that contains the 3.0 kb vector fragment. We did
not do steps 5-7 in the lab exercise).
6. melt the agarose in elution buffer from a company called Qiagen. Pour the
melted agarose with DNA over a DNA affinity matrix column from Qiagen. The
DNA will bind the matrix and the agarose will be washed off. Wash the column.
Elute the DNA with a low-salt buffer.
7. Steps 3-6 are also done on the smaller fragment from the pUC9CAT plasmid
digested with BamH1 and HindIII. This separates the CAT gene from the rest of
the plasmid.
8. Ligate the purified vector and CAT insert fragments as in your lab experiment
9. Transform competent cells with your ligation mix.
10 Select bacteria that have taken up the antibiotic resistance of the vector
(ampicillin resistance) but are not blue on X-gal by plating the transformation
mixture on X-gal Amp plates.
I have put a copy of a gel with the pBluescript plasmid (pBS) and the pUC9CAT
plasmid (pCAT) cut with different enzymes onto a powerpoint show with the
results of the electrophoresis gels done in the lab period. The powerpoint show is
in the cloning lab section and called, Cloning gel results.