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2000/01
Cardiff School of Biosciences
Biochemistry
Level II:
Practical
Manual
Molecular Biology I
Dr Peter Kille
Gene cloning practical manual - Page 1
GENE CLONING PRACTICAL

The purpose of this practical is to introduce you to the basic techniques of gene cloning. The
techniques that you will use include:

The preparation of plasmid DNA
Digestion of DNA with restriction endonucleases
Analysis of DNA fragments by electrophoresis
Ligation of DNA fragments to produce binant molecules
Transformation of bacterial cells
Selection of clones by insertional inactivation of a gene
Selection of clones by expressed phenotypes
Amplification of specific DNA fragments by the polymerase chain reaction (PCR)
Induction of gene expression
Enzyme activity measurement

Particular attention should be paid to the following points:

[1] The manual should be used as a guide to assist you with the experiments rather than as
plete recipe book.
[2] There are certain safety requirements (in addition to those appropriate to normal
laboratory practice) that must be adhered to (see next page). It is a requirement that
you read the section on safety mencing any work.

[3] These experiments will work well but only if the advice on page 4 is followed.
Gene cloning practical manual - Page 2
TIMETABLE

Practicals will be held in lab. 101W every Tuesday. Sessions will start promptly at am
and will continue through until . If you cannot attend any part of the practical please
inform a demonstrator or the lecturer in charge.

Week 1
[1] Preparation of plasmid DNA

[2] Digestio