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.

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.
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.
For restriction digest:

Three diluted restriction enzymes ( Bam HI, Pst I and Eco RI)

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.
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. In the following two pages we have indicated all the links.
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html

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

RISK ASSESSMENT FOR LABORATORY PROCEDURES
GENIE CPD COURSE: CLONE THAT GENE!
Health and Safety information to be announced before the course start
1. Location where the procedure will be carried out
University of Leicester, Adrian Building, Lab MSB 207
2. Brief description of procedures.
All procedures described in detail in course folder;
1: Bacterial plasmid analysis by DNA-transformation of Escherichia coli
2: Plasmid DNA extraction from Escherichia coli
3: Restriction endonuclease digestion of plasmid DNA followed by agarose gel electrophoresis.
3. Identified hazards. i) Micro-organism; E.coli
(Biological hazard): 1, 2 ii) Bunsen burner and methylated spirits (fire hazard): 1 iii) Calcium chloride (irritant chemical hazard): 1 iv) Gel electrophoresis (electrical hazard): 3 v) Sybr Green dye in gel electrophoresis buffer (chemical hazard): 3 vi) UV transilluminator (UV hazard): 3
4. Level of risk: trivial acceptable X moderate significant substantial intolerable
5. Steps to be taken to adequately control the risk.
General: Course participants advised of laboratory safety procedure instructions in course manual.
Course participants required to wear laboratory coats at all time, and gloves when
Specific: handling potential hazardous materials (biologicals and chemicals).
Course participants advised of potential hazardous materials/procedures at the beginning of the course. i) non-pathogenic strains used: course participants advised to follow instructions carefully. ii) course participants instructed to exercise caution. iii) course participants instructed to wear gloves during DNA-transformation procedure. iv) course participants instructed how to connect electrophoresis equipment to power supply. v) course participants instructed to wear gloves electrophoresis procedure. vi) UV dark chamber used.
6. Relevant COSH available for inspection in the laboratory.
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html

If you would like any further advice or assistance relating to this course or other areas of the curriculum concerned with Genetics please contact GENIE (details on front cover of this booklet).
Equipment Loan:

The Wellcome Trust Centre for Human Genetics also has an equipment loan scheme for those able to collect and return kits to the centre : www.well.ox.ac.uk/loan-kits
Equipment/Kit purchase:
The following companies supply equipment¸ kits and reagents specifically for use in schools:

Edvotek: www.edvotek.co.uk

Biorad www.biorad-com ( Biorad Education )

National Centre for Biotechnology Education: www.ncbe.reading.ac.uk

Newton Resources: www.newtonresources.co.uk

Philip Harris http://www.philipharris.co.uk
Separate Reagent Purchase:
Restriction enzymes and DNA ladders, plasmids and competent bacteria etc can also be purchased separately from the following companies who (this is not an exhaustive list):

Life Technologies: www.lifetechnologies.com

New England Biolabs: www.neb.uk.com

Sigma: www.sigmaaldrich.com
Make your own incubator:
 How to make an Incubator for Microbiology: ehow
 Make your own incubator: http://www.umsl.edu/~microbes/pdf/Incubator.pdf
Make your own electrophoresis kit:
 Homemade electrophoresis: www.everything2.com/title/Homemade+electrophoresis
 Building Your Own Tool for Identifying DNA: Forensic Science
Electrophoresis Chamber: www.learn.genetics.utah.edu/content/labs/gel/gelchamber
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html


Where a jumping gene lands on a gene could be important for its effect – o at the end of a gene could be less disastrous, o just before a gene could hit sequences that control the gene expression

This poster only shows 41 genes, but there are 25,000 genes in the human genome. These 41 genes were chosen because they are related to a disease caused by the effects of jumping genes.

Would landing on just one copy of the gene cause the effect, or would both copies have to be disrupted?
1. Teacher and technician notes.docx/rtf/pdf
– this document
2. Student notes.docx/rtf/pdf – document for students to work from
3. Clone that Gene resources.pptx/pdf – the Power Point presentation
4. Setting up restriction digests.doc/rtf/pdf
– this document gives instructions on restriction digests
5. Gel electrophoresis from recombinant clones.doc/rtf/pdf-this document is a guide to gel electrophoresis.
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html