1. 2 enhancing the undergraduate laboratory experience with bio-rad

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  • Enhancing the Undergraduate Laboratory Experience with Bio-Rad

  • Instructors

    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

  • 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

  • How can you fit molecular biology concepts into your curriculum?

  • Majors Course Options: Integrated SeriesCloning and Sequencing

    Protein Expression and Purification

    New Text Book - Biotechnology: A Laboratory Skills Course

  • Cloning and Sequencing Explorer Series

  • Cloning and Sequencing Series

  • 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

  • Techniques Used In Cloning & SequencingStudents 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

  • Large number of species

    Lots of diversity

    Phylogenetic approaches

    Avoid ethical concerns associated with animals

    No pre-approval

    Benefits of using plants

  • What is a Housekeeping Gene? Highly conserved genes that must be continually expressed in all tissues of organisms to maintain essential cellular functions.



    Cytochrome C



  • DNA Extraction Use young, fresh plant-tissue

    DNA extraction at room temperature

    Time requirement ~30 minutes

    Does not require DNA quantification

  • PCR Reactions


    Nested Color-coded PCR primers (hallmark of Bio-Rad PCR kits)

    Two positive controls Arabidopsis pGAP (plasmid DNA)

    One negative control

  • Ligation, Transformation and Plasmid MiniprepsChoose best PCR products for ligation

    Transformation and selection

    Plasmid Miniprep preparation

  • Ligation and TransformationColumn 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)

  • Plasmid minipreps Isolate plasmid DNA (40 minutes)

    Restriction digest (1 hour)

    Electrophorese to confirm inserts (20 minutes)

  • 2.5 kb >2.0 kb >

    1.5 kb >

    1.0 kb >

    0.5 kb >Analysis of plasmid digestspJet vector GAPDH inserts Bgl II Digest

  • 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 ligationInserts can vary from 0.52.5 kb depending on plant speciesDigested and undigested DNA were electrophoresed on a 1% TAE agarose gelpJet vector GAPDH inserts

  • pJet cloning vectorSetting up Sequencing ReactionsAlways need to sequence reverse, complementary strandGAPDH gene of interest

  • 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 sequenceSequencing

  • BioinformaticsTwo month subscription to genetic analysis software from Geospiza

    Data is stored on iFinch server

    Data can be accessed 24/7

  • Students compare 3 sequences

    What is the accurate sequence?

    Usually requires going back to chromatogramsAssembling the full-length contigContiguous sequence

  • Searches a DNA/protein database for published sequences that are similar to your sequenceBasic Local Alignment Search Tool,

    or BLAST BLAST Searches

  • Student Authors

    Great for a resume!

  • Try iFinchhttp://www.geospiza.com/ifinchBioRad.htmlhttp://classroom1.bio-rad.ifinch.com/Finch/

    Username: BR_guestPassword: guestTutorial movies available

  • Protein Expression and Purification Series

  • Protein Expression and Purification Series

  • Protein Expression and Purification Series WorkflowStreak CellsOvernight cultureSubculture, monitor, and induceHarvest and lyse cellsAnalyze

  • Equipment for Separation / Purification16k CentrifugeBioLogic LPBioLogic DuoFlow

  • Protein Expression and Purification Series AdvantagesFollows a complete workflow including bacterial cell culture, induction, fractionation, purification, and analysis of purified proteinTeaches affinity purificationWork with a non-colored protein that is comparable to real world applicationsIncludes ability to run at small scale using a 16k microcentrifuge or scaling up and using chromatography instrumentationPossibility of extensions including western blots, ELISAs, site-directed mutagenesis studies, induction experiments

  • The Value of ProteinsPrice Per GramPrices in 2011 US Dollars* As of 8/14/2011

    Bovine Growth Hormone$14Gold*$56Insulin$60Human Growth Hormone$227,000Granulocyte Colony Stimulating Factor$1,357,000

  • Protein The product of Biotech

    PROTEIN:USED IN THE TREATMENT OF:Cell ProductionInsulinDiabetesE. coliHuman growth hormoneGrowth disordersE. coliGranulocyte colony stimulating factorCancersE. ColiErythropoietinAnemiaCHO cellsTissue plasminogen activatorHeart attackCHO cellsHepatitis B virus vaccineVaccinationYeastHuman papillomavirus vaccineVaccinationYeast

  • 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

  • DHFR and CancerDHFR inhibition or reduction disrupts nucleic acid synthesis affecting-Cell growth-ProliferationMethotrexate chemotherapeutic agent-Competitive inhibitor of DHFR-Methotrexate resistance - correlates with amplification of DHFR genes

  • GST-DHFR-His ConstructGST DHFR - HisGlutathione-s-transferaseAdded to increase solubilityCan be used as a secondary purification methodologyHuman dihydrofolate reductaseGene product of interest Target for chemotherapy reagentsHistidine tag6 Histidine tag that binds to certain metals such as nickel

  • Phases of growth

  • RecoverySeparation of protein from other molecules

    PurificationSeparation of the protein of interest from other proteins

  • Chromatography BasicsMobile 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.

  • His tagsHis tags are typically a series of 6 histidines added to the C or N terminus of a recombinant protein

    HistidineResinNiHis-tagged Recombinant ProteinHis tag and column interaction

  • His tagsImidazoleHistidine His and imidazole structure similarities Imidazole competes with His for Ni2+ sites

  • 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.gifPrinciples of Size Exclusion Chromatography

  • SizeExclusion

  • 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

  • Dont 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

  • 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.comNeed Help? Contact your CTS!

  • CSI and GMO Real-Time PCR

  • 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 s


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