* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Laboratory 2: How do you begin to clone a gene?
Cancer epigenetics wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
Saethre–Chotzen syndrome wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
Non-coding DNA wikipedia , lookup
Neuronal ceroid lipofuscinosis wikipedia , lookup
Genome (book) wikipedia , lookup
Protein moonlighting wikipedia , lookup
Genome evolution wikipedia , lookup
DNA vaccination wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Gene therapy wikipedia , lookup
Gene desert wikipedia , lookup
Gene expression profiling wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Gene expression programming wikipedia , lookup
Epigenomics wikipedia , lookup
Genetic engineering wikipedia , lookup
Point mutation wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Gene nomenclature wikipedia , lookup
Molecular cloning wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Genomic library wikipedia , lookup
Genome editing wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Microevolution wikipedia , lookup
Designer baby wikipedia , lookup
Helitron (biology) wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
History of genetic engineering wikipedia , lookup
LABORATORY 2: HOW DO YOU BEGIN TO CLONE A GENE? LSSI Alum, Shawn Hurley LSSI Alum, Lindsey Engle LSSI Alum, Mary Haus Pre-Assignment http://www.dnaftb.org/34/animation.html • Great background animated storyboard – Could assign as HW if one-toone – Could also complete as pre-lab in computer lab or if you have class sets of tablets or chromebooks – Could complete together in class using teacher computer https://www.youtube.com/watch? v=nfC689ElUVk **Please be aware, sticky ends are not identified correctly, but overall process is. Resource Links/Additional Background https://www.dnalc.org/view/15476-mechanism-of-recombination-3d-animation-with-with-basicnarration.html http://highered.mheducation.com/sites/0072556781/student_view0/chapter14/animation_quiz_ 2.html Important Vocabulary/Terms -Plasmid -Vector -Insert -Construct -Digest -Ligase -Palindromic -Restriction Enzyme -Sticky Ends -Complementary Bases -Origin of replication -Promoter -Antibiotic resistance gene -Regulatory gene Complete Genetic Engineering Sequence Lab 1: Tools of the Trade – Pipetting and Gel Electrophoresis Lab 2: Restriction Digest (cut pKAN-R and pARA) Lab 3: Ligation (combine pieces to make pARA-R) Lab 4: Confirmation (gel electrophoresis) Lab 5: Transformation (introduce pARA-R into bacterial host cells) Lab 6: Culture transformed bacteria, isolate and purify the protein Goals of Lab 2 Logistical (students will coordinate procedural steps necessary to): • Perform restriction digest to generate DNA fragments for future cloning and expression of the red fluorescent protein gene in bacteria Educational (students will be able to): • Identify the common characteristics of plasmids • Explain how plasmids are used as vectors in gene cloning/expression • Describe the function of restriction enzymes • Explain restriction enzymes are used to create recombinant plasmids What is a plasmid? • Comparatively small circular double-stranded DNA molecules of bacterial origin • Range in size from 1,000 to 200,000 base pairs (bp) • Independent of bacterial chromosome, carrying “nonessential” genes Plasmid Features: • Origin of replication • Promoter • Antibiotic resistance • Multiple cloning site (polylinker) • Regulatory element(s) Function: - Region responsible for initiating the copying of plasmid DNA - Site to which RNA polymerase binds to begin transcription - Gene coding for a product that confers antibiotic resistance - Unique restriction sites allow for the digestion of plasmid & introduction of insert (foreign gene) - Gene coding for a product that regulates transcription of the insert promoter regulatory gene MCS (with insert) antibiotic resistance gene origin of replication (Ori) Plasmids as Vectors http://www.pbslearningmedia.org/resource/biot11.sci.life.gen.genengdna/geneticengineering-and-working-with-dna/ What are restriction enzymes? • Catalytic proteins that function like molecular scissors, cutting double-stranded DNA at distinct recognition sites that are usually unique to a particular enzyme. http://highered.mheducation.com/sites/dl/free/0072421975/196644/restriction_endonucl eases.html What are restriction enzymes? Characteristics: • Recognition sites are palindromic sequences, usually 4-8 nucleotides in length 5’ – G A A T T C – 3’ 3’ – C T T A A G – 5’ • Cleave covalent bonds of sugar-phosphate backbone • If enzyme is a staggered cutter, generates sticky ends (unpaired overhangs capable of hydrogen bonding with complementary bases) 5’ – G A A T T C – 3’ 3’ – C T T A A G – 5’ • Nonemclature based on source bacterial species & strain 1st letter roman numeral designates E co R I of genus order of discovery 1st two strain (Escherichia) letters of species (coli) Application of These Molecular Tools – Scientists can build designer plasmids that contain specific restriction sites – This allows scientist to cut out and recombine genes to allow for cloning and gene expression (requires cutting each DNA sample with same restriction enzyme(s)) https://highered.mheducation.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/fr ee/0072437316/120078/bio38.swf::Early%20Genetic%20Engineering%20Experiment Lab 2 – Creating the Digest (cutting up the DNA) • Purpose: to produce the DNA fragments that will be joined to make the recombinant plasmid. – Will need to cut two plasmids • pKAN-R – has the rfp gene with promoter sequence (pBAD) and an antibiotic resistance gene for kanamyacin (kanR) • pARA – has an antibiotic resistance gene for ampicillin (ampR) and a regulatory gene (araC), coding for arabinose activator protein – Arabinose is a sugar that induces transcription of the rfp gene by working with arabinose activator protein – Will use 2 restriction enzymes on each plasmid allowing the segment from pKAN-R to later be inserted into the pARA plasmid • BamHI • HindIII Lab 2 – Creating the Digest Lab 2 – Creating the Digest Restriction Digest Fragments BamH I Hind III 4,706 bp BamH I Hind III 4,496 bp Hind III BamH I 806 bp Hind III BamH I 376 bp Safety-Lab 2 • Use laboratory coats, safety glasses and gloves as appropriate • Avoid restrictive clothing and open-toed shoes • No eating or drinking in the lab • Make sure that students are familiar with the operating instructions and safety precautions before they use any of the lab equipment • Check all MSDS (Material Safety Data Sheets) for all chemicals and reagents in the lab before preparing and running the lab www.amgenbiotechexperience.com Lab & Aliquoting Guide-Lab 2 Reagents/Supplies 1.4 ml 2.5X Buffer/class (2.5xB) Aliquot 20ul/group Storage Temp 4o 110 ul of pKAN/class (pKAN) 10ul/group -20o 110 ul of pARA/ class (pARA) 10ul/group -20o 65 ul of Restriction Enzyme/class (RE) 5ul/group -20o 12mL of DI water/ kit (dH20) 1ml/group RT Equipment/Supplies 10 Student boxes with the following: 1 p20 micropipette 1 p200 micropipette 1 p1000 micropipette 1 waste and 1 ice bucket 4 Mini centrifuges 1 Water bath 2 Floating racks www.amgenbiotechexperience.com 1 microfuge rack 1 bag of microfuge tubes 1 bag of microfuge tubes 1 box of refillable tips (2 ul-200 ul) Notes Keep this for all Labs Completing Lab 2 Teacher Tips – *make sure students label with initials/group *Remind them of mixing techniques *Make sure centrifuge is balanced before running *Identify for students when to use a new tip Completing Lab 2 (pg. B-15 in teacher manual) Label tubes Add 4.0 uL 2.5xB into each Add 4.0 uL K into K+ and K- tubes Add 4.0 uL A into A+ and A- tubes Add 2.0 uL RE into K+ and A+ tubes and mix Add 2.0 uL dH2O in K- and A- tubes and mix Centrifuge all tubes Put all tubes in floating rack and set in 37oC water bath for 60 mins.* Remove and place tubes into freezer overnight * - if time is limited, reducing the digest time to 20-30 minutes is aaceptable Teacher Video Resources • Mixing two solutions video: https://www.amgenbiotechexperience. com/curriculum/curriculumresources/mixing-two-solutions • Digestion video (different digest, but good techniques): https://www.youtube.com/watch?v=Gs Wo8dCivWs • How restriction enzymes work (good, short): https://www.youtube.com/watch?v=lW XryzgRces • Longer, overall of lab 2 created for absent students (screen cast): https://www.youtube.com/watch?v=4 wbStjWEM8A • Fun one from MIT. Covers whole process 1st half good for Labs 2&3: • https://www.youtube.com/watch?v=nf C689ElUVk ***remember, sticky end issue here