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Biotechnology in the NSS Biotechnology in the NSS biology curriculumbiology curriculum
Daniel K. L. Lee, Ph. D.Daniel K. L. Lee, Ph. D.
Department of Applied Biology and Chemical TechnologyDepartment of Applied Biology and Chemical Technology
The Hong Kong Polytechnic UniversityThe Hong Kong Polytechnic University
Rapidly expanding areaUnderstand the principles of the techniquesFamiliarize oneself with the applicationsUse the appropriate instruments and the
proper techniquesPCRGel electrophoresis
The message is
To guide the students through the PCR and gel electrophoresis experiments
with the use of appropriate instruments and proper techniques
so that the students will understand the principles and the applications of these techniques.
To carry out the experiment, we need
The appropriate instrumentsThe necessary reagents and materialsA procedure that worksThe proper techniques
Amplifying DNA using PCR and separating DNA Amplifying DNA using PCR and separating DNA fragments with gel electrophoresisfragments with gel electrophoresis
The instruments
MicropipettesPCR machine (thermal cycler)Gel electrophoresis system
Gel tankGel trayPower supply
Microcentrifuge
InstrumentInstrument Approximate costApproximate cost
Micropipettes HK$800 – 2000
Thermal cycler HK$30,000 upwards
Gel tank + tray HK$2,000 upwards
Power supply HK$5,000 upwards
Microcentrifuge HK$2,000 upwards
•These instruments are available from most biotechnology companies that sell equipments.
•The price of each instrument varies from brand to brand, and also from model to model.
•One needs to strike a balance between functions and cost.•Usually the basic models are good enough for these experiments in secondary schools.
Reagents and materials
Micropipette tipsMicrotubesTemplate DNAPrimersTaq DNA polymeraseDeoxynucleotide triphosphatesPCR bufferAgaroseElectrophoresis bufferGel loading dyeDNA stain
Reagents and materials
Not as expensive as one may thinkNeed to source from different companiesNeed to test and optimize the conditionsMost of the reagents available are mainly
for research purposes, not for secondary school teaching.
For PCR, you need to decide onThe template DNA [human, mouse, plant DNA?]The pair of primers [defines the segment of DNA
to be amplified.]
A procedure that works
Different conditions are required for different DNA samples, different primers, and reagents from different sources.
A procedure that works for human DNA with a specific pair of primers may not work with another pair of primers.
A procedure and a pair of primers that work for human DNA may not work with mouse DNA.
Optimization by test runs required.
Proper techniques
Using the micropipettesUsing the thermal cyclerMixing the reagents in the microtubesUsing the microcentrifugePreparing the agarose gelLoading the samples into the wellsStaining the gel
Proper training for teachers Proper training for teachers and techniciansand technicians
DNA workshops in PolyU
We have undergraduate students who may work as part-time TAs to help you with the preparation, demonstration, and teaching of the experiments.
These are students who have organized the summer DNA camps and workshops, prepared the experiments and taught the students and teachers.
The experiment is done!The experiment is done!ButBut
Learning has just begun!Learning has just begun!
On Gel Electrophoresis
Which direction did the DNA moved, towards the anode or the cathode?
Why is DNA moving in this direction?What is the charge of DNA molecules? Which part of the DNA does the charge come from? [The
phosphate groups along the sugar-phosphate backbone]One phosphate group per base; longer DNA,
more phosphate groups, more charges.Charge/mass ratio is constant for all DNA,
irrespective of length.The mobility solely depends on how easy a DNA
molecule moves through the pores of the gel; smaller DNA easier; larger DNA more difficult.
On the PCR Results
Why is there no band in B and C? [No reagents; no template DNA]
Why do we need to include B and C in the experiment? [Controls]
Why are there two bands in A? two different DNA, of different sizes two different PCR products
Why are there two different PCR products?Two different templates or two different pairs of primersDiploid genome; Heterozygous “Aa” for this locus; basic
genetics concepts.
On the design of the experimentWhy do we need to run the gel?
To separate the DNA fragments by size difference To check if we have the DNA fragment of the correct size To compare between different samples
Why do we need to do PCR? To amplify the DNA To do DNA fingerprinting To detect microbes To detect GM food
What DNA shall we use as template?Which DNA region shall we PCR?What primers shall we use?
This is all about applications.This is all about applications.
Biotech teaching made easy
by packages available from the Life Science Education division of a biotechnology company
Genes in a Bottle Kit [S1, S2]pGLO Bacterial Transformation Kit [S2, S3]Restriction Digestion and Analysis of Lambda
DNA Kit [S4, S5]Crime Scene Investigator PCR Basics Kit
[S5, S6]
