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Enhancing the Undergraduate Laboratory Experience with Bio-Rad

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Bio-Rad Curriculum and Training Specialists:

Sherri Andrews, Ph.D.sherri_andrews@bio-rad.com

Damon Tighe damon_tighe@bio-rad.com

Leigh Brown, M.A. leigh_brown@bio-rad.com

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, Ph.D.sherri_andrews@bio-rad.com

West: Damon Tighedamon_tighe@bio-rad.com

Cental: Leigh Brown, M.A. leigh_brown@bio-rad.com

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