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Enhancing the Undergraduate Laboratory Experience with Bio-Rad
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Bio-Rad Curriculum and Training Specialists:
Sherri Andrews, [email protected]
Damon Tighe [email protected]
Leigh Brown, M.A. [email protected]
Instructors
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Why Use Bio-Rad?
• Guaranteed to work
• Easy to prep
• Cost Effective per student group
• Offer single lab experience or complete research/workflow
• World class technical support
• ISO 9000 certified reagents
• EDU discount
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How can you fit molecular biology concepts into your curriculum?
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Majors Course Options: Integrated Series
Cloning and Sequencing
Protein Expression and Purification
New Text Book - Biotechnology: A Laboratory Skills Course
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Cloning and Sequencing Explorer Series
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Cloning and Sequencing Series
Microbial CulturingAntibiotic SelectionSterile Technique
Genomic DNA ExtractionDNA PrecipitationDNA Quantitation
GAPDH PCRNested PCRDegenerate primersExonuclease
Gel ElectrophoresisDNA Gel InterpretationBand IdentificationStandard Curve Use
CloningDirect PCR cloningTransformationLigation
PCR PurificationSize Exclusion Chromatography
Plasmid MiniprepRestriction Enzyme DigestionGel Electrophoresis
SequencingAutomated sequencing
BioinformaticsSequence Data EditingContig AssemblyIntron-Exon Prediction
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Why TeachCloning & Sequencing Series?
• Students guide the research process and make decisions about their next steps
• Encompasses a myriad of laboratory skills and techniques commonly used in research
• Students generate original data that may lead to publications in GenBank
• Students formulate scientific explanations using data, logic, and evidence
• Students understand research is a process rather than a single experiment giving students a real-life research experience with both its successes and challenges
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Techniques Used In Cloning & Sequencing
Students use the following techniques:
• Micropipetting • DNA extraction• Gel electrophoresis & interpretation• Polymerase chain reaction• DNA purification• Restriction enzyme digests• Microbiological sterile technique• Preparing competent bacteria• DNA ligation• Heat-shock transformation• Plasmid DNA isolation• Sequence analysis• BLAST searching• GenBank submission
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Benefits of using plants
• Large number of species
• Lots of diversity
• Phylogenetic approaches
• Avoid ethical concerns associated with animals
• No pre-approval
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What is a Housekeeping Gene? Highly conserved genes that must be
continually expressed in all tissues of organisms to maintain essential cellular functions.
Examples:
•GAPDH
•Cytochrome C
•ATPase
•ß-actin
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DNA Extraction
• Use young, fresh plant-tissue
• DNA extraction at room temperature
• Time requirement ~30 minutes
• Does not require DNA quantification
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PCR Reactions
Initial
Nested
• Color-coded PCR primers (hallmark of Bio-Rad PCR kits)
• Two positive controls• Arabidopsis• pGAP (plasmid DNA)
• One negative control
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• Choose best PCR products for ligation
• Transformation and selection
• Plasmid Miniprep preparation
Microbial CulturingAntibiotic SelectionSterile Technique Cloning
Direct PCR cloningTransformationLigation
PCR PurificationSize Exclusion Chromatography
Plasmid MiniprepRestriction Enzyme DigestionGel Electrophoresis
Ligation, Transformation and Plasmid Minipreps
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Ligation and Transformation
•Column purification (10 minutes)
• Blunt-end PCR product & ligate to pJet vector (30 minutes)
• Preparation of competent bacteria cells (30 minutes)
• Efficient heat-shock transformation (15 minutes)
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Plasmid minipreps
• Isolate plasmid DNA (40 minutes)
• Restriction digest (1 hour)
• Electrophorese to confirm inserts (20 minutes)
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2.5 kb >2.0 kb >
1.5 kb >
1.0 kb >
0.5 kb >
Analysis of plasmid digests
pJet vector
GAPDH inserts
Bgl II Digest
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Example of a miniprep digestion with Bgl II
Lane 1: 500 bp molecular weight ruler
Lanes 2, 4, 6, 8: minipreps digested with BglII
Lanes 3, 5, 7, 9: undigested minipreps
• Different sizes of inserts suggests different GAPDH genes were cloned in this ligation
• Inserts can vary from 0.5–2.5 kb depending on plant species
1 2 3 4 5 6 7 8 9
Digested and undigested DNA were electrophoresed on a 1% TAE agarose gel
pJet vector
GAPDH inserts
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pJet cloning vector
Setting up Sequencing Reactions
Always need to sequence reverse, complementary strand
GAPDH gene of interest
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• Sanger method of sequencing
• 4 fluorescent dyes- 1 for each base
• DNA fragments separated by CE
• Fragments separated in sequential order
• iFinch screens out low quality sequence
Sequencing
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Bioinformatics
• Two month subscription to genetic analysis software from Geospiza
• Data is stored on iFinch server
• Data can be accessed 24/7
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• Students compare 3 sequences
• What is the accurate sequence?
• Usually requires going back to chromatograms
Assembling the full-length contig
Contiguous sequence
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Searches a DNA/protein database for published sequences that are similar to your sequence
Basic Local Alignment Search Tool,
or BLAST
BLAST Searches
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Student Authors
Great for a resume!
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Try iFinch
http://www.geospiza.com/ifinchBioRad.html
http://classroom1.bio-rad.ifinch.com/Finch/
Username: BR_guest
Password: guest
Tutorial movies available
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Protein Expression and Purification Series
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Protein Expression and Purification Series
Option 1CentrifugationPurificationModuleOption 3
PrepackedCartridgePurificationModule Option 2
HandpackedColumnPurificationModule
Growth andExpressionModule
SDS-PAGEElectrophoresisModule
DHFREnzymaticAssayModule
PurificationModule
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Protein Expression and Purification Series Workflow
Streak Cells
Overnight culture
Subculture, monitor, and induce
Harvest and lyse cells
Purify
Centrifugation or Instrumentation
Analyze
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Equipment for Separation / Purification
16k Centrifuge BioLogic LP BioLogic DuoFlow
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Protein Expression and Purification Series Advantages
• Follows a complete workflow including bacterial cell culture, induction, fractionation, purification, and analysis of purified protein
• Teaches affinity purification
• Work with a non-colored protein that is comparable to real world applications
• Includes ability to run at small scale using a 16k microcentrifuge or scaling up and using chromatography instrumentation
• Possibility of extensions including western blots, ELISAs, site-directed mutagenesis studies, induction experiments
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The Value of Proteins
Bovine Growth Hormone $14
Gold* $56
Insulin $60
Human Growth Hormone $227,000
Granulocyte Colony Stimulating Factor
$1,357,000
Price Per Gram
Prices in 2011 US Dollars* As of 8/14/2011
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PROTEIN: USED IN THE TREATMENT OF:
Cell Production
Insulin Diabetes E. coli
Human growth hormone Growth disorders E. coli
Granulocyte colony stimulating factor Cancers E. ColiErythropoietin Anemia CHO cellsTissue plasminogen activator Heart attack CHO cellsHepatitis B virus vaccine Vaccination YeastHuman papillomavirus vaccine Vaccination Yeast
Protein – The product of Biotech
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DHFR —Dihydrofolatereductase
•Converts dihydrofolate into tetrahydrofolate (THF) by the addition of a hydride from NADPH
•THF is a methyl (CH3) group shuttle required for synthesis of essential molecules
- nucleotides- amino acids
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DHFR and Cancer
•DHFR inhibition or reduction disrupts nucleic acid synthesis affecting
-Cell growth-Proliferation
•Methotrexate – chemotherapeutic agent-Competitive inhibitor of DHFR-Methotrexate resistance - correlates with
amplification of DHFR genes
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GST-DHFR-His Construct
GST – DHFR - His
Glutathione-s-transferase
•Added to increase solubility
•Can be used as a secondary purification methodology
Human dihydrofolate reductase
•Gene product of interest
•Target for chemotherapy reagents
Histidine tag
•6 Histidine tag that binds to certain metals such as nickel
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Phases of growth
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Recovery
Separation of protein from other molecules
Purification
Separation of the protein of interest from other proteins
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Chromatography Basics
• Mobile phase (solvent and the molecules to be separated)
• Stationary phase (through which the mobile phase travels)– paper (in paper chromatography)– glass, resin, or ceramic beads (in column
chromatography)
• Molecules travel through the stationary phase at different rates because of their chemistry.
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His tags
N3H+-OOC
Histidine
Resin
• His tags are typically a series of 6 histidines added to the C or N terminus of a recombinant protein
Ni
Ni
Ni
Ni
N
NH
NN
H His-tagged Recombinant
Protein
• His tag and column interaction
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His tags
Imidazole
N3H+-OOC
Histidine
• His and imidazole structure similarities• Imidazole competes with His for Ni2+ sites
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• Beads in column are made of polyacrylamide and have tiny pores
• The mixture of molecules is added to the column
• Large molecules move through the column quickly traveling around the beads
• Smaller molecules move through the pores of the beads and take longer to pass through the column
http://tainano.com/Molecular%20Biology%20Glossary.files/image047.gif
Principles of Size Exclusion Chromatography
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SizeExclusion
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Protein Analysis • Determination of success of induction, lysis, and purification of GST-DHFR-His using SDS-PAGE analysis
• Measurement of concentration using the absorbance at 280 nm
• Enzymatic activity analysis
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Don’t take our word for it!
• Patty Aune – NC State University – 40 t0 50 lab sections every spring use pGLO, GFP Purification, and Forensic DNA Fingerprinting
• University of Virginia – Use Cloning and Sequencing for 10 lab sections
• Joann Lau - Bellarmie University – Use Cloning and Sequencing and Protein Expression and Purification
• Gordon Wells - Ohio University – All kits
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Curriculum Training Specialists
East: Sherri Andrews, [email protected]
West: Damon [email protected]
Cental: Leigh Brown, M.A. [email protected]
Need Help? Contact your CTS!
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CSI and GMO Real-Time PCR
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What is Real-Time PCR?
•The Polymerase Chain Reaction (PCR) is a process for the amplification of specific fragments of DNA.
•Real-Time PCR a specialized technique that allows a PCR reaction to be visualized “in real time” as the reaction progresses.
•Real-Time PCR allows us to measure minute amounts of DNA sequences in a sample!
•Teach melt curve analysis and serial dilution
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Genes in a Bottle
Students extract and preserve their own DNA
LIFE SCIENCE CONCEPTS & SKILLS:
Cellular structures– membrane, organelles
DNA– location, solubility– genetic information– extraction from tissue
Enzyme properties & function– use of protease to aid in extraction
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pGLO™ Bacterial Transformation
Students transfer a jellyfish gene to bacteria
Can regulate the expression of GFP
LIFE SCIENCE CONCEPTS & SKILLS:Central Dogma of Molecular Biology
– DNA>RNA>Protein>Trait– Genetic engineering
Prokaryotic cells– Cell biology, genetic transmission– Antibiotic resistance
Microbiology techniques– Sterile technique– Growth media
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Chromatography Kits:
GFP Chromatographypurify the bioluminescent protein (extension to pGLO)
Secrets of the Rainforest™study the ethical issues of biotechnology
Size Exclusion ChromatographySeparate vitamin B12 and hemoglobin using chromatography
LIFE SCIENCE CONCEPTS & SKILLS:Chromatography
– Protein properties– Tools for protein purification
• Creating a commercial product– Scientific, ecological, ethical and legal issues of
biotechnology
Biomolecules– Physiological function– Naturally pigmented molecules– Maintenance of deleterious alleles (e.g. sickle cell
anemia)
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Microbes and Health – “What Causes Yogurtness?”
Isolate the microorganisms from yogurt and inoculate milk, linking the “sick” milk to the causative microbe
LIFE SCIENCE CONCEPTS & SKILLS:
Isolate yogurt-causing bacteria– Follow Koch’s postulates
– Microbiology and human disease
– Biotechnology and food production
Learn laboratory microbiology skills– Study food microbiology and bacterial metabolism
Inquiry Based– Do different brands
of yogurt contain different
microbes?
– Directly linked to students
daily lives
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Forensic DNA Fingerprinting
Compare Crime Scene DNA with 5 Suspects to determine “Who Done it?”
Restriction Analysis of Lambda DNA
Use restriction enzymes and gel electrophoresis to analyze DNA
LIFE SCIENCE CONCEPTS & SKILLS:Allelic differences in populations
– DNA as a unique identifier
– Heredity
Restriction enzymes– Function and properties
– Origin in bacteria and use in genetic engineering
– Plasmid mapping
Agarose Gel Electrophoresis– Separation of molecules using
electrical current
– Size based on distance traveled
– Creating buffers
Micropipetting techniques
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Crime Scene Investigator PCR Basics™
Students learn to use the polymerase chain reaction (PCR) and DNA electrophoresis to determine the genotypes of 5 DNA samples
Simulate real-world crime lab techniques using one of the Short Tandem Repeat (STR) loci commonly used in forensic typing.
LIFE SCIENCE CONCEPTS & SKILLS:Allelic differences in populations
– DNA as a unique identifier– Use of PCR in DNA profiling– Polymorphic loci and multiple alleles– Non-coding DNA sequences
Agarose Gel Electrophoresis– Separation of molecules using electrical current– Size based on distance
traveled– Creating buffers
Micropipetting techniques
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PV92 PCR Informatics
Students isolate and examine the DNA fingerprint of their own DNA
LIFE SCIENCE CONCEPTS & SKILLS:Population Genetics
– Hardy Weinberg equilibrium
– Evolution, migration, speciation
– Bioinformatics using
Cold Spring Harbor allele server
Polymerase Chain Reaction– Uses in forensics, archeology, research, disease
diagnostics
Agarose Gel Electrophoresis– Separation of molecules using
electrical current
– Size based on distance traveled
– Creating buffers
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GMO Investigator™
Students extract and amplify DNA from grocery store foods
LIFE SCIENCE CONCEPTS & SKILLS:
Agriculture and environment– Pesticides and herbicides
– Population growth and
environmental challenges
– Plant biodiversity and ecosystems
Molecular Biology– DNA replication and PCR
– Genetic transformation to create GMOs
– Control of gene expression
(in foreign hosts)
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Got Protein?™ Kit
Determine the protein concentration in common beverages such as milk, sports drinks and more
Engages students – they bring in their samples of choice
LIFE SCIENCE CONCEPTS & SKILLS:
Proteins – Essential molecules in all living cells
Bradford Assay– Coomassie Blue dye binds to protein and allows
for quantitation and detection
– correlation between the amount of blue color and the amount of protein
Spectrometry– Use of spectrophotometer
– Apply Beer’s Law
Inquiry based - which foods/drinks provide more proteins?– Directly links to students daily lives
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ELISA Immuno Explorer™
Students simulate spreading an infectious disease and detect origin
LIFE SCIENCE CONCEPTS & SKILLS:
Immunology– antigen-antibody interaction
– virology
– infectious diseases
Enzyme Properties– enzyme-substrate interaction
– colorimetric detection
Real world applications– Pregnancy, drug, HIV testing
– GMO and environmental testing
– bioterrorism
Types of ELISA Tests included:• Tracking the spread of a
disease (epidemiology)• Detecting specific
antigens in a sample• Diagnosing past exposure
to a disease
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Comparative Proteomics Kits:
Protein Profiler – Module
Evolution Wet Lab – create protein profiles for different fish species
LIFE SCIENCE CONCEPTS & SKILLS:Molecular evolution
– Natural selection and genetic diversity
– classification
Polyacrylamide Gel Electrophoresis– protein separation techniques
Module 1
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Comparative Proteomics Kits:
Western Blot – Module
Takes Protein Profiler to the Next Level – identify the specific protein using antibodies
LIFE SCIENCE CONCEPTS & SKILLS:Molecular evolution
– Natural selection and genetic diversity
– classification
Western blotting– Explore immunodetection
– Protein separation techniques
– Antibodies as tools
Module 2
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Biofuel Enzyme Kit LIFE SCIENCE CONCEPTS & SKILLS:
• Enables both qualitative and quantitative measurements of reactions
- Determine the rate of reaction in the presence or absence of an enzyme
- Determine the effect of temperature, pH, enzyme concentration, substrate concentration on the rate of reaction
- Test the ability of mushroom extracts to increase the rate of reaction
Guides instruction on enzyme kinetics and biofuel energy sources
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Webinars • Enzyme Kinetics — A Biofuels Case Study
• Real-Time PCR — What You Need To Know and Why You Should Teach It!
• Proteins — Where DNA Takes on Form and Function
• From plants to sequence: a six week college biology lab course
• From singleplex to multiplex: making the most out of your realtime experiments
explorer.bio-rad.comSupportWebinars