cape biology workshop on concepts in biotechnology & genetic engineering prepared and presented...
TRANSCRIPT
CAPE Biology Workshop on Concepts in Biotechnology
& Genetic EngineeringPrepared and presented by
Dr. Marcia E. RoyeEmail: [email protected]
Senior Lecturer in BiotechnologyAssociate Dean for Graduate Studies and Research, FST
Office: Biotechnology Centre, ground floor Tel: 927-0304 (Office)
935-8518/9, 977-1828 or ext. 2518-20 (Centre)
Useful Documents at Dr Roye’s Bulletin Board
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Objectives of Today’s Lab Experiment
•Schedule of today’s activity•Nucleic acid: Overview extraction•Introduction to plasmids•Introduction to restriction enzymes•Laboratory procedure
Schedule of Activities•Host: The Biotechnology Centre and, UWI, Mona.•Venue: The Faculty of Medical Sciences Teaching and Research Complex
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
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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.
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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.).
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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
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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?
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Restriction Enzymes movie
•Restriction enzymes recognize specific sequences in DNA and cleaves the DNA at these specific sequences.
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Restriction Enzymes Pst1•Restriction enzymes are names based on the bacteria they are isolated from e.g.
OrganismRestriction
enzyme Recognition sequence
Provendica Stuartii Pst1
5'CTGCA↓G 3' 3'G↓ACGTC 5'
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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.
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Agarose Gel
Experiment 1
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
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.]
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.
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.
Restriction Digestion mix
•Your plasmid 5.0 µl •Digestion buffer 1.5 µl •Water 7.5 µl •Pst1 1.0 µl •Total 15 µl
•Escherichia
