1. Email: marcia.roye@uwimona.edu.jm
CAPE Biology Workshop on Concepts in Biotechnology & Genetic Engineering
Prepared and presented by
Dr. Marcia E. Roye
Senior Lecturer in Biotechnology
Associate Dean for Graduate Studies and Research, FST
Office: Biotechnology Centre, ground floor
Tel: (Office)
/9, or ext (Centre)

2. Dr Roye’s Bulletin Board

3. Useful Documents at Dr Roye’s Bulletin Board

4. Objectives of Today’s Lab Experiment
Schedule of today’s activity
Nucleic acid: Overview extraction
Introduction to plasmids
Introduction to restriction enzymes
Laboratory procedure

5. Schedule of Activities
Host: The Biotechnology Centre and, UWI, Mona.
Venue: The Faculty of Medical Sciences Teaching and Research Complex

6. Schedule of Activities
8:30-9:00 am Registration
9:00-11:00 am DNA extraction
11:00-12:00 pm Restriction Digestion of DNA
12:00- 1:00 pm Lunch break
1:00- 2:00 pm Agarose Gel Electrophoresis of restricted DNA
2:00-4:00 pm Lab discussion: Interpretation of Results

7. Isolation of Nucleic Acid
Where in the cell is nucleic acid found?
To extract DNA/RNA from cells:
Break plasma and nuclear membranes (NaOH and SDS).
Cell contents will leak out into the solution.
In solution (proteins, nucleic acids, lipids, carbohydrates, etc.)
Separate the other macromolecules from the nucleic acids.
The main problems are:
the removal of protein
removal of RNA during DNA preparation
removal of DNA during RNA preparation.

8. Removal of proteins Proteins are removed by:
Proteases (protease K) or a detergent eg. Phenol.
Proteins can also be extracted with high salt concentration e.g. potassium acetate.
RNA can be removed with RNAse and DNA can be removed by DNAse.
The nucleic acid is precipitated using ethanol ( 2 vol.) or isopropanol (1 vol.).

9. Plasmids: pCR 2.1 or pTrc99a
Plasmids are small genetic elements which replicate independently of the chromosome.
Most plasmids are double stranded DNA and are circular.
They are found in most bacteria and many have more than one plasmid.
Plasmids have genes that confer important properties to the cell e.g.
antibiotic resistance
degradation of unusual material in the environment

10. Restriction Enzymes
This makes it possible for molecular biologists to isolate, modify and move genes from one organism or place in an organism to another.
The discovery of restriction enzymes was the beginning of what is know as genetic engineering today.
Do you know of any genetically engineered organism?

11. Restriction Enzymes movie
Restriction enzymes recognize specific sequences in DNA and cleaves the DNA at these specific sequences.

12. Restriction Enzymes Pst1
Restriction enzymes are names based on the bacteria they are isolated from e.g.
Organism
Restriction enzyme
Recognition sequence
Provendica Stuartii
Pst1
5'CTGCA↓G 3' 3'G↓ACGTC 5'

13. Gel Electrophoresis movie
A piece of DNA can be cut with a restriction enzyme and the resulting fragments can be separated by agarose gel electrophoresis.
DNA molecules of different size can be separated based on their size.
DNA molecules are –vely charged and will move towards the +ve electrode when placed in an electric field.
Therefore restriction analysis and gel electrophoresis allows us to separated DNA fragments of different sizes.

14. Agarose Gel

15. Experiment 1

16. Methodology
To pellet cells, spin down 1.5 ml of culture in a microfuge tube for 3 minutes at 10,000rpm and discard the supernatant. This is already done for you
Completely resuspend the pellet in 100 µl of Solution 1.
Add 200 µl Solution 2, mix by gently inverting about 6 times and incubate at room temperature for 5 minutes.
Add 150 µl Solution 3, mix by flicking the tube

17. Methodology
Centrifuge at 10,000 rpm for 10 min and transfer as much of the supernatant as possible to a new labeled tube, avoiding as much as possible the white precipitate. (Discard tube with white precipitate).
Add 240 µl of isopropanol, mix thoroughly (by gently inverting 4-6 times) and centrifuge at 10,000 rpm for 10 min.
Remove the supernatant from tubes by pipetting [Be careful: the white pellet contains your plasmid DNA. Try not to dislodge the pellet.]

18. Methodology Wash the DNA pellet by adding 500 µl of 70% ethanol.
Spin for 5 min at 10,000 rpm and remove by pipetting as much of the ethanol as possible.
Dry pellets [laboratory assistants will collect your tubes for the drying].
Dissolve dried pellets in 30 µl TE/RNase. 

19. Cutting DNA with Pst1
Give each group a tube with 10 µl of Pst1 and buffer
You will need to add 5 µl of your DNA
Incubation at 37°C until you get back from lunch
Load DNA on agarose gel to separate DNA fragments.

20. Restriction Digestion mix
Your plasmid 5.0 µl
Digestion buffer 1.5 µl
Water µl
Pst µl
Total µl

21. Escherichia