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BIO230 Lab notes.pdf

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Kenneth Yip

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BIO230LabNotes Lab1-RapidIsolationandElectrophoresisofRNAfromE.coli • rRNAisthemostabundantandstableformofRNAinmostcells(70-80%) • MostoftheRNAdetectedfromgelelectrophoresisisrRNAandsometRNA • Thereare3speciesofprokaryoticrRNA:23S,16S,and5S.(smallernumber=smallerRNA) Chemicalsinvolved: • EDTA-achelator.ThisisusedtoprotectyourRNAfrombeingdigested.Ithasanaffinityforcationssuch asMg2+.Likewise,RNAseshaveanaffinityfortheseionsaswell.EDTAessentiallytakesmostofthe availableMg2+leavingRNAsesunabletobindtothem.RNAsesarethenunabletoactivate,andhence cannotdegradetheRNA. • Lysozyme-usedtobreakdownbacterialcellwallsbyhydrolyzingthelinkagesbetweentheir polysacchardies.Allowsbetteraccesstocell. • SDS-adetergent.Itsolubilizestheplasmamembraneanddenaturesproteins.SDSiswatersoluable. • PotassiumAcetate-usedforprecipitation.Isinsolubleinwater,soboundwithSDSitcanbeusedtotakeall themoleculesthathavebeenboundtoSDSandprecipitatethemfromtheaqueousenvironment.Withthe useofthismixedwithSDSandacetrifuge,proteins,largemolecules,andcellfragmentscanberemovedto leaveRNAandsmallDNA. • NaCl-neutralizesnegativebackboneofRNA.UsedwithethanoltoprecipitateRNA • Ethanol-tocounteractthehighsaltconcentrationadded.RNAneedstobeprecipitatedfromthesolution,so ethanolisusedbecauseithasahigheraffinityforbindingtowaterthanRNA.PrecipitatestheRNAfromthe solution • SYBRsafe-astain.EmitsgreenlightwhenirradiatedwithUV.UsefulforvisualizingRNAandDNAingels. Ispossiblycarcinogenic Electrophoresis: • Fragmentsizeisbiggestdeterminingfactorforhowfastitmovesongel.Smallerfragmentsmovemore. • Svedbergunitsareameasureofrateofmovement(denotedbyS)onthegravitationalfield.Thesmaller theS,thefastertherateofmovement.(Hence5SisthesmallestrRNAandmovesthemostingel) BIO230LabNotes Lab2/3-GeneRegulation • Generegulationcantakemanyformsandoccurinanumberofpathways • Themostcommonformofregulationinbacteriaistranscriptional pGLOproperties: • itisanengineeredplasmidforfluorescence,ampicilinresistance,andanaraCoperon • PBADisthepromoterregion.WhenboundtoRNApolymerase,theothergenesonpGLOaremade • GFP-greenfluorescentprotein.Thisisupstreamofthepromoter.IfRNApolymeraseisbound,thesample shouldfluoresce bla-beta-lactamaseenzyme.Thisencodesforresistancetoampicillinantibiotic.Thepurposeofthisisto • makesurethattheplasmidactuallyhastheGFPandaraC.Ifblaisn’tpresent,neitheraretheothers,and thebacteriawilldiebecauseitisinanampicillindish. • araC-downstreamofpromoter.BindingsiteforaraCiscalledara1.Presenceofhigharabinosewillactivate twounitsofaraCiintoaraCawhichwillbindtoara1.WithtwoaraCboundtoara1,theRNApolymerasecan sticktothepromoter. • CBS-Capbindingsequence.Thisrespondstolevelsofglucose.Withlowlevelsofglucose,cAMPcanbind intoCAPitomakeitCAP3(activemode).CAP3cannowbindtoCBSandallowforRNApolymerasetobind tothepromoter. • Overall,lowglucoseandhigharabinoseisneededtoallowRNApolymerasetopositiononthepromoterand synthesizethegenes.ThiswillsynthesizeblaandGFP,allowingthebacteriatofluoresceandsurvivein ampicillin. Transformations: • AtransformationisinsertingforeignDNAintoacell(bacterialinthiscase) plasmidsareusedbecausetheyareindependentofchromosomesandabundantinbacterialcells,so • engineeringthemiseasier. • Movingtheplasmidintothehostcellrequiresheatshockandaninfluxofcalciumcations. • Antibioticsareusedbecausenotalltransformationswork,soonlytransformedbacteriasurvivebecause theyhaveantibioticresistance. transformationefficiency: #ofcoloniesonplate/ngofplatedDNA X1000ng #ofcoloniesisusuallyagiven.Onecolonystemsfromonebacteriatypically. platedDNAcalculation: [concentrationofDNAusedtotransformxvolumeofDNAaddedtomix]x[volumeactuallyplatedon transformationmix/volumeoftotaltransformationmix] totalcalculation: #coloniesonplate -------------------------------------------------------------------------------------------- =transformants/ug ([DNAused]xvolumeDNAadded)xvolumeactuallypl------------------------------ volumeoftotalmix BIO230LabNotes Lab 4 Notes * One of the most neglected concepts is proper configuration of the microscope with regard to illumination. Optimizing illumination is the key to enhanced microscope performance. * Light emitted from various locations on the lamp filament be collected and focused at the plane of the condenser aperture diaphragm is very important. * In plant cells, parts of the cytoplasm may show cytoplasmic streaming, which may result in broad cytoplasmic strands that transiently traverse the vacuole. Individual organelles such as the mitochondria, will exhibit short periods of rapid abrupt movements with or against the cytoplasmic flow. * There is a layer of cytoplasmic and organelles close to the PM that does not take part in the cytoplasmic streaming of the cytoplasm. * The nucleus maintains its position in cell regardless of cytoplasmic movements around it. * Fluorescein diacetate (FDA) is an uncharged and non-fluorescent derivate of the fluorescent molecule-fluorescein. Because FDA is uncharged, it passively crosses the PM but once inside the cell, cell esterases convert FDA to fluorescent, polar molecule. * Fluorescein cannot easily diffuse back across the plasma membrane escape from the cell. * Dead cells that lack esterase activity or have a damaged PM do not retain fluorescein. * Therefore, FDA is commonly used as a method to distinguish live or dead cells. * Fluorescein fluoresces a green or yellow colour when irradiated with blue light. (green or yellow depends on pH). * The use of genetically coded fluroescent proteins (e.g. GFP) has allowed us to tag and track an enormous range of proteins in space and real time in their natural environment without the application of exogenous probes or fixation of cells and tissues that can produce artifacts. * Magnification: an apparent increase in size. * Resolution: the resolving power being the ability to distinguish between two very closely positioned objects. * l.r.= (0.61lamda)/N.A. * The limit of resolution (I.r.) is related to the shape of the cone of light entering the objective lens. * N.A.= cone or numerical aperture * Lamda=wavelength * The larger the N.A., the greater the resolving power. * The smaller the wavelengths of light, the greater the resolving power. BIO230LabNotes * Brownian motion is vibrating particles in cell, this means cell is dead or dying. Article * GFP was first discovered as a companion protein to aequorin, the chemiluminescent protein from A. Victoria. * GFP by itself was found to fluoresce under excitation, requiring no substrate or coenzymes. GFP contained all of the information needed for proper synthesis of the fluorophore. * S65T= was found to accelerate the speed of fluorophore formation. * Some mutations helped the molecule to fold correctly at 37 degrees. * The protein expression was improved by converting wild-type GFP condons to form enhanced GFP (EGFP). This produced brighter molecules. EGFP can be seen in low light intensities and little photo-bleaching. * Developments of GFP provided variants with differing absorbance and emission spectra, allowing the simultaneous visualization of distinct GFP variants in a cell. * BFP= blue. Used for multicolor imaging and FRET ( fluorescence energy transfer) * CFP =cyan. B/w BFP and EGFP * YEP= red-shifted yellow. Brighter than CFP. * CFP –YEP has been used to study protein-protein FRET * 4D microscopy= time-lapse observations of fluorescent molecules are collected as 3D data rather than 1 image in a single focal plane. * FRAP= fluorescence recovering after photobleaching. An area of the cell is photobleached with a high intensity laser and the movem
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