Clone that Gene
Student Notes
Background Information
This is a practical activity is for teachers and technicians to gain an appreciation of how
gene cloning is carried out, but also the importance of gene cloning in todays world.
Why clone genes?
Scientists clone genes for several reasons, for example
 to study genes; in order to learn more about them.
 to use the genetic information to make products, e.g. insulin.
 for screening purposes, to determine whether an individual has a gene linked to
disease.
If a scientist is cloning a gene, it means that he or she is isolating a gene from all the other
genes of that organism and making multiple copies of it. One way to do this is to use a
plasmid vector. Plasmids are small circular DNA molecules often found in bacteria, but
which are separate to the main bacterial chromosome. Fragments of DNA from another
source can be inserted into these plasmids. The resulting recombinant molecules can then
be transferred into bacteria, and as the bacteria divide they copy the recombinant
molecules thus propagating the inserted segments of DNA. Since each bacterium only
takes up a single recombinant plasmid molecule, as it divides and grows into a colony all
its progeny will also contain this recombinant molecule. A collection of DNA clones each
carrying a different DNA fragment from the organism of interest is called a gene library.
Once a gene library is made, scientists may use a range of molecular techniques to find
the gene of interest, literally "finding the needle in a haystack". The recombinant plasmid
can then be used for further investigation.
The molecular techniques you will be carrying out during this course are based around a
simple gene cloning experiment. It is not possible to literally clone a gene in a day,
however, the experiment we have designed incorporates techniques and approaches that
are central to gene cloning, and can easily be reproduced within the classroom.
1) The Activity
The aim of the experiment: is to distinguish which sample is which. In order to determine
this you will be performing physical analysis of the DNA as well as finding the phenotypes
conveyed by the plasmids.
2) Materials
For restriction digest:
 Three diluted restriction enzymes (BamHI, PstI and EcoRI)
 10x reaction buffer
 Sterile water
 Plasmid DNA
Gel electrophoresis:
 The gels for this step have been prepared for you in advance. If you would like
further information on how to make and run gels A GENIE educational video will be
shown, explaining how to make and run a DNA agarose gel. This video is also
viewable on-line on the Virtual Genetics Education Centre a link to this video can
be found in the information on the provided USB memory drive.
E.coli transformation:
 Two DNA samples (plasmid 1 and plasmid 2), on ice
 Three eppendorf tubes containing E.coli culture (25μl each tube) on ice
 Sterile water, on ice
 1 Luria agar plate containing tetracycline (labelled LT)
 1 Luria agar plate containing ampicillin (labelled LA)
 1 Luria agar plate containing kanamycin (labelled LK)
 Luria broth (labelled LB)
 Beaker of Chloros disinfectant. (WARNING, CORROSIVE)
Analysis of transformants
 You are provided with agar plates from a previous phenotype analysis experiment.
3) Procedure
Participants should be provided with two samples of DNA.
 One of the samples should contain a plasmid vector.
 The second DNA sample contains a recombinant plasmid, which consists of the
original vector with an extra piece of DNA added.
This hands-on experience you will gain during this course will provide you with a deeper
understanding of the subject and techniques involved. Many of these molecular techniques
now form a major part of the A-level curricula.
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html
Links in the curricula
DNA structure:

AQA AS/A2 GCE Biology 2410 – Version 1.4:
Unit 2, p.11-18: 3.2.2

AQA AS/A2 GCE Human Biology 2405 – version 1.3 :
Unit 2, p.9-16: 3.2.1

EDEXCEL AS/A2 GCE Biology 9BI01– Issue 4
Unit 1, p21-26 Topic 2.

OCR AS/A2 GCE Biology H021 H421- version 3:
AS Unit F212: Module 1 p.12-15– 2.1.2

WJEC AS/A2 GCE Biology 2009
AS Assessment Unit BY1 p.11-13, 1.6
Restriction digest, restriction enzymes and the production of sticky ends:

AQA AS/A2 GCE Biology 2410 – Version 1.4.:
Unit 5, p.27-32: 3.5.8

AQA AS/A2 GCE Human Biology 2405 – version 1.3:
Unit 4, p.18-24: 3.4.4

OCR AS/A2 GCE Biology:
AS Unit F215: Module 2 p40-42 – 5.2.3

WJEC AS/A2 GCE Biology 2009:
AS Assessment Unit BY5 p.20-23, 5.6
Recombinant DNA and genetically modified organisms:

AQA AS/A2 GCE Biology 2410 – Version 1.4.:
Unit 5, p.27-32: 3.5.8

AQA AS/A2 GCE Human Biology 2405 – version 1.3:
Unit 4, p.18-24: 3.4.4

EDEXCEL AS/A2 GCE Biology 9BI01– Issue 4
Unit 5, p.39-44 Topic 8.

OCR AS/A2 GCE Biology:
AS Unit F215: Module 2 p.40.-42, 5.2.3

WJEC AS/A2 GCE Biology 2009:
AS Assessment Unit BY5 p.20-23, 5.6
Gene cloning and transfer:
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html

AQA AS/A2 GCE Biology 2410 – Version 1.4.:
Unit 5, p.27-32: 3.5.8

AQA AS/A2 GCE Human Biology 2405 – version 1.3:
Unit 4, p.18-24: 3.4.4

WJEC AS/A2 GCE Biology 2009:
A2 Assessment Unit BY5 p.20-23, 5.6
Ligase:
 AQA AS/A2 GCE Biology 2410 – Version 1.4.:
Unit 5, p.27-32: 3.5.8

AQA AS/A2 GCE Human Biology 2405 – version 1.3:
Unit 4, p.18-24: 3.4.4
Plasmids:
 AQA AS/A2 GCE Biology 2410 – Version 1.4.:
Unit 3, p.5-10: 3.1.3

OCR AS/A2 GCE Biology:
AS Unit F215: Module 2 p.40-42, 5.2.3

WJEC AS/A2 GCE Biology 2009:
A2 Assessment Unit BY5 p.20-23, 5.6
Gel electrophoresis:

AQA AS/A2 GCE Biology 2410 – Version 1.4.:
Unit 5, p.27-32: 3.5.8

EDEXCEL AS/A2 GCE Biology 9BI01 – Issue 4
Unit 4, p34-38 Topic 6.

OCR AS/A2 GCE Biology:
AS Unit F215: Module 2 p.40-42, 5.2.3
The ethics of cloning:
 AQA AS/A2 GCE Biology 2410 – Version 1.4.:
Unit 5, p.27-32: 3.5.8

AQA AS/A2 GCE Human Biology 2405 – version 1.3:
Unit 4, p.18-24: 3.4.4

EDEXCEL AS/A2 GCE Biology 9BI01– Issue 4
Unit 5, p.39-44 Topic 8.

OCR AS/A2 GCE Biology:
AS Unit F215: Module 2 p.40-42, 5.2.3

WJEC AS/A2 GCE Biology:
A2 Assessment Unit BY5 p.20-23, 5.6
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html