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WHAT YOU NEED TO KNOW ABO U T W O RKI N G IN A

WHAT YOU NEED TO KNOW ABO U T W O RKI N G IN A. LAB- GENE R AL L A B OR G ANIZ A TION. D r . Fer n T sien Depart m ent o f Genetics LSU H SC. Wha t typ e o f laborator y do yo u wor k in?. In gener a l, labs ca n be d e scribed as:

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WHAT YOU NEED TO KNOW ABO U T W O RKI N G IN A

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  1. WHATYOUNEEDTOKNOW ABOUT WORKING INA LAB- GENERALLABORGANIZATION Dr.FernTsien DepartmentofGenetics LSUHSC

  2. Whattypeoflaboratorydo youworkin? • Ingeneral, labscanbedescribedas: • BasicScience–the researchersdo sciencefor the sakeof knowledge.Forexample,howdoesa specific transcription proteinwork? • Clinicalresearch–humanpatients or their samples are usedto investigatea diseaseor syndrome.

  3. Whereisyourlablocated? • Eachlabispartofa DepartmentorCenter. • Large piecesofequipmentareoftensharedbythe department membersand arehoused inaseparatelabor possiblyyourlab. • Thesecan includeultra-cenrifuges, liquid • nitrogentank,autoclaves,darkrooms, etc. • Alsothedepartmentmay haveaconference room andakitchen. • Makesure youcheckif you can usethesefacilitiesbefore goingahead.

  4. KNOWYOURLABCOWORKERS

  5. PrincipalInvestigator(PI) • Peopleoftenask:Who is yourPI? • Thisisthe headof the lab, the boss,your • advisor.Mostprobablyyourmentor. • Thispersonoftenspendstimeintheir officewriting grantsor researchreports andmayspendlesstimeinthe lab. • Iftheyare clinicians, theymayalso see • patientsinadditionto reportwriting. • However,this personisthe intellectual guidebehindmostof theprojectsinthe lab • PIsare responsibleforfundingthelab research.

  6. •I I

  7. Postdocs • LabPost-doctoral fellow • Thispersonhas recentlyreceivedtheirdoctorate(usuallyPhD)andisdoingatrainingperiodbefore becoming aPI • Theyusuallywork ontheirownproject, butwillcollaboratewith otherlab membersonvariousparts oftheproject.

  8. Technicianorresearch assistant • Canbeacollegestudentwho wantsto gainmoreexperienceinalabbefore enteringmedicalorgraduateschool • Canbe aprofessionalwithamasters degreeandappropriatepayandtitle, basedonyearsofexperience • Theydoavarietyoftasksincluding: orderinglabsupplies,preparingmedia,caringfor thelab’scelllines,assistingthelabwithexperiments,and theycancarry theirownexperiments.

  9. Labsupervisor • The day-to-dayoperationofthelabis • sometimesoverseenby a labsupervisor. • Thisperson usually hasamastersdegreeand extensivelabexperience.

  10. Graduatestudents • Theyaredoinglabworktoget • theirMastersdegreeor PhD. • Theyusuallyworklong hours andspenda lotof timeinvestedin theirspecificproject. • Theybecome increasingly independent duringtheir4-5 yearsinthelab.

  11. Residentsandfellows • They havean MDandarespendingsometime(weeks,months,sometimesyears) traininginan areaof his/herfieldbeforethey becomeindependent

  12. Workinghours • Becauseexperimentsdonotfitintoaslotof9-5,lab workershaveunpredictableandsometimeseccentric hours.Thisdoesnotapplytoyou! • Clinicalresearchalsodependsontheclinicschedule. • Checkwithyourmentorwhatworkinghoursare • expectedofyou andtryto conformtothis. • Pleasecommunicatewithyourmentorandlab personnel whatyourvacation plansare. • If youaresick,pleaseletthemknow. • Even thoughyoudonotgetpaidbythehour,youneed toletthemknowoutofcourtesy.

  13. Labmeetings • Largelabsusuallyhavemeetingsto discusstheresearchof • eachmemberofthe team. • Inthesemeetings,sometimesonly one or two peopleare assignedtotalkeachweek, orallmembersare expected to speakbriefly. • Somemeetingsare casual, and someareformalwitha slide • projector. • Youmaybe askedto participate.Ask yourmentorahead of timeif he/she expectsyouto participate so youcan prepare afew statementsorquestionsyou mayhave. • Check out theatmosphereandaskquestionsduring appropriatetimes. • DO:take notesandlookuptermsyou maynot • understand • DONOT:fallasleep,text messageduringthemeeting

  14. Thefirstweek • Bynow youshouldhavebeenassignedadesk • orlab bench. • Your mentor shouldhave discussedyour projectwithyou. • Ifanother labmember offers to teach you, go • aheadandlearnfromthem. • Youmayget alabkey.Becareful withit! • Yourresearchmaybe slowrightnow while youwaitforreagents tobeordered,cellsto grow,etc.

  15. Whattodoonthe firstfewweeks • Setupandorganizeyourlab benchor desk • Introduceyourselfto everyone in thelab if the mentorhas notdoneso. • TAKENOTESON EVERYTHING!Youdo not wantyour instructorsto keeprepeatingthemselvesortheywillget annoyed. • Familiarizeyourselfwith where thingsare kept. • ASK.Ofcourse,youdo not want tobotheranyone unnecessarily,but itis muchbetter to ask about a procedure,a reagent,equipment,etc. than towastedtime andmoney. • Ifyoumakea mistake,it’sOK.Justmake sure youlearnand • askcoworkersforhelp. • Askyourmentorforrelevantliterature abouttheproject.Thiswillimpressthem!

  16. WhatNOTtodoonyour firstfewweeks • Don’tsay“we did not do it thisway inmyprevious job”. Eachlab does experimentstheir ownway (example:cellculture) • Donot propupyour feetandread,playcomputergames, takeanap,or lookat videos onYoutube.Itistruethatat thebeginning,there willbe somedead(non-experiment)time untilthe experimenttakes off. However, playingwhileothersare workinghardwillnot makea goodimpression. Insteadtryto readabout yourresearch topic. • Donotaskand/orcomplainto otherstudents or co-workersaboutmoney • orsalaries.Itcanirritate mentors andotherco-workers. • Don’tsayyouare workingina labfor anyother reasonthanfor interestin the field. Ifyousay youare workingtherejustfor the money, you willnot betakenseriouslyanditcancostyouyourjob.

  17. Basicsurvivalrules: Attitude • Ask,donot command.Othersare takingthe trouble tohelpyouanddo not HAVEto doit. • Becourteoustoeveryone,not just your mentor. • Donot assumethatsomeoneis goingto dropwhat • theyare doingto helpyou.Bepatient. • Writedowneverythingwhensomeoneisgiving you instructions.You arenot expectedto remember everything,andthat person is not expectedto repeateverythingoverand overagain. • Makeappointmentswithbusy people.Some mentorsarealso directorsof adepartmentor center, andare hardto knowwhenthey are available for themto focus on whatyouhaveto say.

  18. Basicsurvival rules:Courtesyat ScienceFriction thelabbench • Donot use reagentsinsomeoneelse’slab bench without asking. • Ifeveryonesharesa reagent andyoufinishit,let someoneknow.Donot placethe emptybottleback on theshelf. • Ifsomethingbreaks,tellsomeoneimmediately.You will notgetintroubleunlessyou tryto hideit. • Ifyoudo somethingwrong,confess.Everyonemakes mistakes,but sneakingto covera mistakeis unacceptable. • Clean up aftertheexperiment.Donot leaveit for the technicianor graduatestudent todoit.

  19. Checkyourlab’sownSafety rules • No eating,drinking,orsmokingin thelabunless • approvedbyyourmentor. • Do notwearopentoedshoesin thelab.Yourfeet willbe vulnerable ifachemicalspillsonyou. • No shortsorbaremidriffsareallowedforsafety • reasons. • Do notmouthpipet,evenwater. • Askwhattodoincaseofemergency-wherethefirstaidkitiskept,howtocallforemergency

  20. Ifyouhaveanyquestionsor • problems,pleasemakesuretolet usknowearlyonintheinternship. Dr. Paula Gregory Dr.FernTsien pgrego@lsuhsc.edufmille@lsuhsc.edu

  21. TODAY’S EXPERIMENT

  22. WhatareHeLaCells? • HeLacellsarecervicalcancercellsthatwere • obtainedformawomannamedHenriettaLacks. • These cellswereobtainedfromabiopsyofthe cervixbyherdoctorwithouther knowledge/consent. • Thecellswereimmortalized. Henrietta Lacks

  23. ImmortalizedcellsandHeLa • HeLacellsobtainedfromthebiopsyarestillalive and havebeenusefulin theunderstandingof canceranddisease,aswellasdiscovery of treatmentsand therapy. • HeLawasthefirstcelllineimmortalized • Was usedin thedevelopmentofpoliovaccine, andtofindlinkbetweenhumanpapillomavirus (HPV)andcervicalcancer

  24. TheImmortalLifeofHenrietta Lacks • byRebeccaSkloot • Littlewasknow about HenriettaLacksuntilrecently. • Recently,RSkloot wrote thebiography ofMs.Lacks: • PoorAfricanAmerican womanfromBaltimore • Had5children • Hadcervicalcancer • Had littleunderstandingofher diagnosisofcervical cancer,andnoknowledgeofwhatthedoctoratJohns Hopkins wasplanningto do withherbiopsy. • Died ofcancer in 1951lessthan a yearafterdiagnosis

  25. Ethicalimplications • Most widelyused andpropagatedcelltypeusedfor • research. • Billionsofdollarsintreatmentandtherapydiscoveriesby researchersandpharmaceutical companies • Lastyear, theNationalInstitutesofHealth (NIH)madeanagreementwiththeLacksfamily • The Lacks family agreedtoallowresearchersto have limitedaccessto her DNA sequence • Lacksfamilyhas controloverher information

  26. Newregulations • Currently,therearespecificregulationsfor ensuringpatientconfidentialityandtoallow patientstogive consenttousetheircellsfor research. • Cellsarenotallowedtobeusedforresearch • withoutthepatientconsent.

  27. HeLainthenews • “The HeLaGenome:AnAgreementonPrivacy • andAccess”;StatementreleasedbytheNIH • HBOtofilmmoviebyOprahWinfrey

  28. Today’sexperiments: • Learn howto micropipette • HeLaand normalDNA • PCRanalysisforthedetectionof human papillomavirus(HPV) • Gelelectrophoresis

  29. WhatisPCR? • Thepolymerasechainreaction(PCR)isa fasttechniqueused inmanylabsto "amplify“orcopysmallsegmentsofDNA. • Itisone of the mostimportantscientificadvancesinmolecular • biology. • Itscreator,KaryB.Mullis,wasawarded theNobel Prize for Chemistryin 1993.

  30. WhatisPCRusedfor? • Onceamplified,theDNAproducedby PCR(PCR product)canbeusedinmanydifferent laboratory procedures. • DNAfingerprinting inforensicsandpaternity testing • Detectionofbacteria or viruses(HIV/AIDS) • Diagnosis ofgeneticdisorders • Many types of research!

  31. PCR:PolymeraseChainReaction • Usedto makemillionsofexactcopiesof • DNAfroma biologicalsample • Allowsverysmallsamplesto beanalyzed, • suchasasampleofa fewskincells • Mustbe verycarefulabout contaminationin • thisprocess

  32. InsidethePCR reactiontube: • DNAthat youwant to • analyze • Primers(littlepiecesofDNAthat define thearea thatyouwant to analyze) • Nucleotides(A,T,C,G) • Taq polymerase(drivesthis • entire reaction) • Magnesium, salts,etc.(for solutionstability) • Sterile distilledwater

  33. Millionsof DNAcopies 45°C 72°C 95°C ThePCR machineheatsandcoolstheDNA,primers,andotherreagentsin thetubeandallows theamplificationto takeplace. This resultsinmillionsofcopies.

  34. 1.Restrjctionenzymescleave DNAintosmaJmersegments ofvarioussizes. 2.DNAsegmentsare loadedintowellsina porous gel.Thegelfloatsin abuffersolutionwithinachamber betweentwoelectrodes_ 3.wnenanelectriccurrentispassedthroughthechamber. EBDNAfragmentsmovetoward tileposilively--cilargedcalhode. 111 I III II 4.SmallIerDNIAsegments movefasterandfa.riheir thanlargerDNAsegments_

  35. VariationsofPCRandgel electrophoresis • RT-PCR • Q-PCR • Western blots • Northern • blots • Etc….

  36. THEMICROPIPETTE

  37. warningsaboutgel electrophoresis: • THEPOWERSUPPLYGENERATESANELECTRIC CURRENT.DONOTIMMERSEYOURHANDINTHE BUFFER OFTHEGELAPPARATUSWHILETHEPOWER SUPPLYISCONNECTED ANDTURNEDON. • UVLIGHTDAMAGESEYES.MAKESURETOKEEP THEUVTRANSILLUMINATORLIDDOWNTOLOOKAT GEL.MAKESURENOTTOOPENTHELIDWHILETHE UVLIGHTISTURNEDON.

  38. Gelelectrophoresis • Remove thegel combsanddams. • Arrangegelonitsgel tray intothe gel electrophoresis apparatus, making sure that thenegativeside(black)is onthesidewiththewells. • Addenoughelectrophoresis bufferto submerge thegel.

  39. Gelelectrophoresis • Retrievethe PCR reactiontubesfrom the PCR machineandmicrocentrifuge5 seconds • Toeachtubeadd5 µl of loadingdyetoeachsample. Mix bypipettingup and down. • Loadgelby pipetting10 ulof thesolutioninto each • ofthe wells. • Writedownthe orderinwhichyouloadedthe • samples.

  40. Gelelectrophoresis • Attachthecovertotheelectrophoresis apparatusmatchingthecolor codedcablesand electrodes(e.g.,redtoredandblacktoblack). • Connectthecolorcodedcablestothepowersupply.Turnthepower supply“ON”andsetthe voltageto“100V.”Press“RUN.” • Lookforbubblestomakesureitisworking • properly. • Letsamplesuntiltheloadingdyehasmoved about¾ofthewaydownthegel(abouthalfanhour). • Dr.Gregory’spresentation

  41. Gelelectrophoresis • Turn off thepower supply andremovecover of • electrophoresisapparatus. • Wearinggloves,pickup the gel andtrayand drain theliquidinto the electrophoresisapparatus. • Carefullytransferthegelon its trayto theUV • transilluminator.Turnoff theroomlights. • MAKESURETHE LIDTOTHETRANSILLUMINATOR ISCLOSEDand then turnon theUVtransilluminator. Viewthe gel andphotographusing a cellphone camera. • Turn off theUVtransilluminator,discardthe gelina wastebagprovidedbyyour teacherand wipe the transilluminatorwith papertowels.

  42. TypicalPCR Results 300bp 200bp

  43. Housekeeping • EATINGandDRINKINGarenot permittedin labsthatcontain biological,chemicalorradiological materials • Disposeoftrashin theappropriate containerwhenitis generatedto preventaccumulation • Donotplaceemptybottlesor otherhazardsin walkways • Keepchemicalsandglassware awayfromtheedgesof countertops • Immediatelycleanupanyspills GoodHousekeeping PoorHousekeeping

  44. FireSafety • Knowthelocationofthenearestfire extinguisherandemergencyexits • Duringa fire,usestairs,notelevators • Flamecabinetisrequiredforstorageof 10Gal.ormoreofanyflammable substance • Compressedgascylindersmustbe secured • Electricalcordsshouldbefreeof • breaksorfraying • Intheeventofanalarm: • Secureanymaterialthatleft unattendedwouldposeahazard • Exitproperly Acceptable Unacceptable Acceptable Unacceptable

  45. EyewashandShowers • Knowwherethenearesteyewashandemergencyshower stationsare • Ifyou aren’tsureordon’tknowhowtousethem,askyour • supervisor

  46. HazardInformation • Beawareofhazardlabelsonentrydoorsandonthe doors • ofrefrigerators,freezers,microwavesanddewars • YoushouldbewearingthecorrectPersonalProtective Equipment(PPE)beforeopeninganydoorwitha hazard labelonit • MSDS–MaterialSafetyDataSheets • Includechemicalandphysicalproperties • Toxicityandhealtheffects • Compatibility,safehandlingandstorage • Spillandfireresponse

  47. Anagentofbiological originthathasthecapacitytoproduce deleteriouseffectsin humans,suchasmicroorganisms,toxinsand allergensderivedfromthosemicroorganisms,andallergensand toxinsderivedfromhigherplantsandanimals. • Examples Bacteria Fungi Parasites Toxins Viruses Prions Rickettsials

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