1 / 94

Working in a Lab: Organization and Key Insights

Discover the different types of laboratories and how they operate, learn about lab co-workers and their roles, understand working hours and lab meetings, and get tips for your first few weeks in the lab.

ywilhelm
Download Presentation

Working in a Lab: Organization and Key Insights

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related