Cov txheej txheem kuaj pom molecular muaj peev xwm tsim tau ntau cov nucleic acid los ntawm kev ua kom cov khoom me me pom hauv cov qauv. Txawm hais tias qhov no muaj txiaj ntsig zoo rau kev ua kom pom kev rhiab heev, nws kuj qhia txog qhov ua tau ntawm kev ua qias tuaj los ntawm kev nthuav dav ntawm cov tshuaj aerosols hauv chav kuaj mob. Thaum ua cov kev sim, cov kev ntsuas tuaj yeem ua kom tsis txhob muaj kev ua qias tuaj ntawm cov tshuaj reagents, cov khoom siv hauv chav kuaj mob thiab qhov chaw zaum, vim tias kev ua qias tuaj yeem tsim cov txiaj ntsig cuav (lossis cuav-tsis zoo).
Yuav kom txo tau qhov yuav muaj kev sib kis kab mob, yuav tsum ua raws li Kev Xyaum Ua Haujlwm Zoo hauv Chaw Kuaj Mob txhua lub sijhawm. Tshwj xeeb, yuav tsum ceev faj txog cov ntsiab lus hauv qab no:
1. Kev siv cov tshuaj reagents
2. Kev npaj chaw ua haujlwm thiab cov khoom siv
3. Kev siv thiab kev ntxuav cov lus qhia rau qhov chaw molecular uas tau teev tseg
4. Cov lus qhia dav dav txog kev kawm txog molecular biology
5. Kev tswj sab hauv
6. Cov Ntawv Sau
1. Kev siv cov tshuaj reagents
Ua ntej qhib, siv lub raj centrifuge reagent ua ntej kom tsis txhob muaj cov aerosols. Muab cov reagents faib ua ob feem kom tsis txhob khov thiab ua rau cov khoom siv tsis huv. Sau daim ntawv lo thiab hnub tim rau txhua lub raj reagent thiab cov tshuaj tiv thaiv thiab khaws cov ntaub ntawv teev cov reagent thiab cov lej batch uas siv rau hauv txhua qhov kev sim. Siv lub raj lim dej los lim tag nrho cov reagents thiab cov qauv. Ua ntej yuav, nws yog ib qho tseem ceeb kom paub tseeb nrog tus neeg tsim khoom tias cov lim dej haum rau hom pipette uas yuav siv.
2. Kev npaj chaw ua haujlwm thiab cov khoom siv
Yuav tsum teeb tsa qhov chaw ua haujlwm kom ntseeg tau tias qhov kev ua haujlwm tshwm sim hauv ib qho kev taw qhia, los ntawm thaj chaw huv (ua ntej PCR) mus rau thaj chaw qias neeg (tom qab PCR). Cov kev ceev faj dav dav hauv qab no yuav pab txo qhov muaj feem yuav kis tau kab mob. Muaj chav sib cais, lossis tsawg kawg yog thaj chaw sib cais, rau: kev npaj mastermix, kev rho tawm nucleic acid thiab kev ntxiv DNA template, kev nthuav dav thiab kev tuav cov khoom nthuav dav, thiab kev tshuaj xyuas cov khoom, piv txwv li gel electrophoresis.
Hauv qee qhov chaw, muaj 4 chav sib cais yog qhov nyuaj. Ib qho kev xaiv uas ua tau tab sis tsis xav tau yog ua qhov kev npaj mastermix hauv thaj chaw kaw, piv txwv li lub txee laminar flow. Hauv qhov xwm txheej ntawm nested PCR amplification, kev npaj mastermix rau qhov kev tshuaj ntsuam thib ob yuav tsum tau npaj rau hauv thaj chaw 'huv' rau kev npaj mastermix, tab sis kev txhaj tshuaj nrog cov khoom PCR thawj zaug yuav tsum tau ua hauv chav amplification, thiab yog tias ua tau hauv thaj chaw kaw tshwj xeeb (piv txwv li lub txee laminar flow).
Txhua chav/cheeb tsam xav tau ib pawg sib cais ntawm cov pipettes, cov taub lim dej, cov tube racks, vortexes, centrifuges (yog tias muaj), cov cwj mem, cov tshuaj reagents dav dav, cov tsho tiv no hauv chav kuaj thiab cov thawv hnab looj tes uas yuav nyob ntawm lawv qhov chaw ua haujlwm. Yuav tsum ntxuav tes thiab hloov hnab looj tes thiab cov tsho tiv no hauv chav kuaj thaum txav mus los ntawm thaj chaw uas tau teev tseg. Cov tshuaj reagents thiab cov khoom siv yuav tsum tsis txhob tsiv ntawm thaj chaw qias neeg mus rau thaj chaw huv. Yog tias muaj qhov xwm txheej hnyav tshwm sim uas cov tshuaj reagents lossis ib daim khoom siv yuav tsum tau tsiv rov qab, nws yuav tsum tau ntxuav nrog 10% sodium hypochlorite ua ntej, tom qab ntawd so nrog dej huv.
Lus Cim
Cov tshuaj sodium hypochlorite 10% yuav tsum tau ua kom tshiab txhua hnub. Thaum siv rau kev ntxuav cov kab mob, yuav tsum ua raws li lub sijhawm sib cuag tsawg kawg yog 10 feeb.
Los yog, cov khoom lag luam uas tau lees paub tias yog cov tshuaj tua kab mob DNA uas ua rau puas tsuaj tuaj yeem siv tau yog tias cov lus qhia kev nyab xeeb hauv zos tsis pub siv sodium hypochlorite lossis yog tias sodium hypochlorite tsis haum rau kev tua kab mob ntawm cov khoom siv hlau.
Qhov zoo tshaj plaws, cov neeg ua haujlwm yuav tsum ua raws li txoj cai ua haujlwm ib txoj kev thiab tsis txhob mus ntawm thaj chaw qias neeg (tom qab PCR) rov qab mus rau thaj chaw huv (ua ntej PCR) rau tib hnub. Txawm li cas los xij, tej zaum yuav muaj qee lub sijhawm uas qhov no tsis zam tau. Thaum muaj lub sijhawm zoo li no tshwm sim, cov neeg ua haujlwm yuav tsum ceev faj ntxuav tes kom huv si, hloov hnab looj tes, siv lub tsho tiv no hauv chav kuaj mob thiab tsis txhob coj cov khoom siv uas lawv xav coj tawm ntawm chav dua, xws li phau ntawv kuaj mob. Cov kev ntsuas tswj hwm no yuav tsum tau hais txog hauv kev cob qhia cov neeg ua haujlwm txog cov txheej txheem molecular.
Tom qab siv tas, yuav tsum ntxuav cov chaw zaum nrog 10% sodium hypochlorite (ua raws li dej huv kom tshem tawm cov tshuaj dawb seem), 70% ethanol, lossis cov tshuaj tua kab mob DNA uas tau lees paub tias muaj nyob hauv khw. Qhov zoo tshaj plaws, yuav tsum teeb tsa cov teeb ultra-violet (UV) kom tshem tawm cov kab mob los ntawm kev siv hluav taws xob. Txawm li cas los xij, kev siv cov teeb UV yuav tsum txwv rau cov chaw ua haujlwm kaw, piv txwv li cov txee kev nyab xeeb, txhawm rau txwv tsis pub cov neeg ua haujlwm hauv chav kuaj raug UV. Thov ua raws li cov lus qhia ntawm lub tuam txhab tsim khoom rau kev saib xyuas teeb UV, qhov cua thiab kev ntxuav kom ntseeg tau tias cov teeb tseem ua haujlwm tau zoo.
Yog siv 70% ethanol es tsis siv sodium hypochlorite, yuav tsum tau siv lub teeb UV los ntxuav kom huv si.
Tsis txhob siv sodium hypochlorite los ntxuav lub vortex thiab centrifuge; hloov ntawd, so nrog 70% ethanol thiab tso rau hauv lub teeb UV, lossis siv cov tshuaj tua kab mob DNA uas muag hauv khw. Yog tias muaj kev nchuav, nug tus neeg tsim khoom kom paub ntxiv txog kev ntxuav. Yog tias cov lus qhia ntawm tus neeg tsim khoom tso cai, cov pipettes yuav tsum tau ntxuav kom huv si los ntawm autoclave. Yog tias cov pipettes tsis tuaj yeem ntxuav tau, nws yuav tsum txaus los ntxuav lawv nrog 10% sodium hypochlorite (ua raws li kev so kom huv si nrog dej huv) lossis nrog cov tshuaj tua kab mob DNA uas muag hauv khw thiab tom qab ntawd raug UV.
Kev ntxuav nrog cov sodium hypochlorite uas muaj feem pua ntau yuav ua rau cov yas thiab cov hlau puas tsuaj yog tias ua tas li; xyuas cov lus pom zoo los ntawm cov chaw tsim khoom ua ntej. Txhua yam khoom siv yuav tsum tau calibrate tsis tu ncua raws li lub sijhawm teem tseg uas cov chaw tsim khoom pom zoo. Ib tus neeg raug xaiv yuav tsum yog tus saib xyuas kom ntseeg tau tias lub sijhawm calibration raug ua raws li, cov ntaub ntawv teev tseg tau khaws cia, thiab cov ntawv qhia kev pabcuam tau tso tawm kom meej meej rau ntawm cov khoom siv.
3. Kev siv thiab kev ntxuav cov lus qhia rau qhov chaw molecular uas tau teev tseg
Pre-PCR: Kev npaj cov tshuaj reagent aliquoting / mastermix: Qhov no yuav tsum yog qhov chaw huv tshaj plaws ntawm txhua qhov chaw siv rau kev npaj cov kev sim molecular thiab zoo tagnrho yuav tsum yog lub txee laminar flow uas muaj lub teeb UV. Cov qauv, cov kua qaub nucleic acid uas tau rho tawm thiab cov khoom PCR uas tau amplified yuav tsum tsis txhob raug tuav hauv thaj chaw no. Cov tshuaj amplification reagents yuav tsum khaws cia rau hauv lub tub yees (lossis lub tub yees, raws li cov lus pom zoo ntawm cov chaw tsim khoom) hauv tib qho chaw uas tau teem tseg, zoo tagnrho nyob ib sab ntawm lub txee laminar flow lossis thaj chaw pre-PCR. Cov hnab looj tes yuav tsum tau hloov txhua zaus thaum nkag mus rau thaj chaw pre-PCR lossis lub txee laminar flow.
Yuav tsum ntxuav thaj chaw ua ntej PCR lossis lub txee laminar flow ua ntej thiab tom qab siv raws li nram no: So tag nrho cov khoom hauv lub txee, piv txwv li pipettes, tip boxes, vortex, centrifuge, tube racks, pens, thiab lwm yam nrog 70% ethanol lossis tshuaj tua kab mob DNA, thiab cia kom qhuav. Yog tias thaj chaw ua haujlwm kaw, piv txwv li lub txee laminar flow, muab lub hood tso rau hauv lub teeb UV rau 30 feeb.
Lus Cim
Tsis txhob cia cov tshuaj reagents raug lub teeb UV; tsuas yog tsiv lawv mus rau hauv lub txee thaum nws huv si. Yog tias ua qhov rov qab sau PCR, nws kuj tseem yuav pab tau kom so cov chaw thiab cov khoom siv nrog cov tshuaj uas rhuav tshem RNases thaum kov. Qhov no yuav pab kom tsis txhob muaj cov txiaj ntsig tsis tseeb los ntawm kev puas tsuaj ntawm enzyme ntawm RNA. Tom qab tshem tawm cov kab mob thiab ua ntej npaj cov mastermix, cov hnab looj tes yuav tsum tau hloov dua ib zaug, thiab tom qab ntawd lub txee npaj siv.
Pre-PCR: Kev rho tawm/ntxiv cov qauv nucleic acid:
Yuav tsum tau rho tawm cov nucleic acid thiab ua haujlwm hauv thaj chaw thib ob uas tau teem tseg, siv cov pipettes sib cais, cov taub lim dej, cov txee raj, cov hnab looj tes tshiab, cov tsho tiv no hauv chaw kuaj mob thiab lwm yam khoom siv. Thaj chaw no kuj yog rau kev ntxiv cov qauv, kev tswj hwm thiab cov kab sib txawv rau cov raj mastermix lossis cov phaj. Txhawm rau kom tsis txhob muaj kev ua qias tuaj ntawm cov qauv nucleic acid uas tau rho tawm uas tau raug soj ntsuam, nws raug pom zoo kom hloov cov hnab looj tes ua ntej ua haujlwm cov kev tswj hwm zoo lossis cov qauv thiab siv cov pipettes sib cais. PCR reagents thiab cov khoom amplified yuav tsum tsis txhob raug pipetted hauv thaj chaw no. Cov qauv yuav tsum tau khaws cia rau hauv cov tub yees lossis cov tub yees khov uas tau teem tseg hauv tib thaj chaw. Qhov chaw ua haujlwm qauv yuav tsum tau ntxuav tib yam li qhov chaw mastermix.
Tom Qab-PCR: Kev nthuav dav thiab kev tswj hwm cov khoom nthuav dav
Qhov chaw tshwj xeeb no yog rau cov txheej txheem tom qab ua kom nrov dua thiab yuav tsum sib cais ntawm thaj chaw ua ntej PCR. Feem ntau nws muaj cov thermocyclers thiab cov platforms tiag tiag, thiab qhov zoo tshaj plaws yuav tsum muaj lub txee laminar flow rau kev ntxiv cov khoom PCR puag ncig 1 rau qhov kev tshuaj tiv thaiv puag ncig 2, yog tias nested PCR tab tom ua. PCR reagents thiab cov nucleic acid uas tau rho tawm yuav tsum tsis txhob raug tuav hauv thaj chaw no vim tias qhov kev pheej hmoo ntawm kev ua qias tuaj siab heev. Thaj chaw no yuav tsum muaj ib txheej hnab looj tes sib cais, tsho tiv no hauv chav kuaj, phaj thiab raj khib, pipettes, cov taub lim dej, cov thoob khib nyiab thiab lwm yam khoom siv. Cov raj yuav tsum tau centrifuged ua ntej qhib. Qhov chaw ua haujlwm qauv yuav tsum tau ntxuav tib yam li qhov chaw mastermix.
Tom Qab PCR: Kev Tshuaj Xyuas Khoom
Chav no yog rau cov khoom siv nrhiav khoom, piv txwv li cov thoob gel electrophoresis, cov pob hluav taws xob, UV transilluminator thiab lub kaw lus sau ntawv gel. Thaj chaw no yuav tsum muaj cov hnab looj tes sib cais, tsho tiv no kuaj mob, cov tais thiab lub raj khib, cov pipettes, cov taub lim dej, cov thoob khib thiab lwm yam khoom siv. Tsis pub coj lwm yam tshuaj reagents tuaj rau hauv thaj chaw no, tsis suav nrog cov xim pleev xim, cov cim molecular thiab agarose gel, thiab cov khoom buffer. Qhov chaw ua haujlwm kuaj mob yuav tsum tau ntxuav tib yam li qhov chaw mastermix.
Lus tseem ceeb
Qhov zoo tshaj plaws, cov chav ua ntej PCR yuav tsum tsis txhob nkag mus rau tib hnub yog tias twb tau ua haujlwm hauv cov chav tom qab PCR lawm. Yog tias qhov no tsis zam tau kiag li, xyuas kom meej tias ntxuav tes kom huv si thiab hnav cov tsho tiv no hauv chav kuaj mob. Cov phau ntawv kuaj mob thiab cov ntaub ntawv yuav tsum tsis txhob coj mus rau hauv cov chav ua ntej PCR yog tias lawv tau siv hauv cov chav tom qab PCR; yog tias tsim nyog, luam cov ntawv luam tawm ntawm cov txheej txheem / cov qauv ID, thiab lwm yam.
4. Cov lus qhia dav dav txog kev kawm txog molecular biology
Siv cov hnab looj tes tsis muaj hmoov kom tsis txhob muaj kev cuam tshuam rau kev sim. Kev siv pipetting kom raug yog qhov tseem ceeb rau kev txo qhov muaj kuab paug. Kev siv pipetting tsis raug tuaj yeem ua rau muaj kev txaws thaum faib cov kua thiab tsim cov aerosols. Kev xyaum zoo rau kev siv pipetting kom raug tuaj yeem pom ntawm cov txuas hauv qab no: Gilson phau ntawv qhia rau pipetting, Anachem pipetting cov yeeb yaj kiab, Centrifuge raj ua ntej qhib, thiab qhib lawv kom zoo zoo kom tsis txhob muaj kev txaws. Kaw cov raj tam sim ntawd tom qab siv kom tsis txhob muaj cov kuab paug.
Thaum ua ntau yam kev tshuaj tiv thaiv, npaj ib qho mastermix uas muaj cov tshuaj reagents sib xws (piv txwv li dej, dNTPs, buffer, primers thiab enzyme) kom txo qhov ntau ntawm cov tshuaj reagent hloov pauv thiab txo qhov kev hem thawj ntawm kev ua qias tuaj. Nws raug pom zoo kom teeb tsa mastermix rau ntawm dej khov lossis lub thaiv txias. Kev siv cov enzyme Hot Start yuav pab txo qhov kev tsim cov khoom tsis tshwj xeeb. Tiv thaiv cov reagents uas muaj cov tshuaj fluorescent probes ntawm lub teeb kom tsis txhob puas tsuaj.
5. Kev tswj sab hauv
Suav nrog cov kev tswj hwm zoo thiab tsis zoo uas tau piav qhia zoo, lees paub tias muaj tseeb, nrog rau kev tswj hwm tsis muaj qauv hauv txhua qhov kev tshuaj tiv thaiv, thiab ntau lub ntsiab lus titrated trendline rau kev tshuaj tiv thaiv ntau. Kev tswj hwm zoo yuav tsum tsis txhob muaj zog heev uas nws ua rau muaj kev pheej hmoo ntawm kev ua qias tuaj. Suav nrog cov kev tswj hwm rho tawm zoo thiab tsis zoo thaum ua kev rho tawm nucleic acid.
Nws raug pom zoo kom muab cov lus qhia meej tso rau hauv txhua qhov chaw kom cov neeg siv paub txog cov cai ntawm kev coj cwj pwm. Cov chaw kuaj mob uas pom cov DNA lossis RNA qis heev hauv cov qauv kuaj mob yuav xav siv txoj kev ruaj ntseg ntxiv ntawm kev muaj cov tshuab tswj huab cua sib cais nrog lub zog cua zoo me ntsis hauv chav ua ntej PCR thiab lub zog cua tsis zoo me ntsis hauv chav tom qab PCR.
Thaum kawg, kev tsim kom muaj ib txoj kev npaj ua kom zoo (QA) yuav pab tau. Ib txoj kev npaj zoo li no yuav tsum muaj cov npe ntawm cov tshuaj reagent master stocks thiab cov khoom siv ua haujlwm, cov cai rau kev khaws cov khoom siv thiab cov tshuaj reagents, kev tshaj tawm cov txiaj ntsig ntawm kev tswj hwm, cov kev pab cuam cob qhia cov neeg ua haujlwm, cov txheej txheem daws teeb meem, thiab cov kev kho thaum xav tau.
6. Cov Ntawv Sau
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Mifflin T. Teeb tsa lub chaw kuaj PCR. Cold Spring Harb Protoc. 2007;7.
Schroeder S 2013. Kev saib xyuas tsis tu ncua ntawm cov centrifuges: kev ntxuav, kev saib xyuas thiab kev tua kab mob ntawm centrifuges, rotors thiab adapters (Daim ntawv dawb No. 14). Hamburg: Eppendorf; 2013.
Viana RV, Wallis CL. Kev Xyaum Ua Haujlwm Hauv Chaw Kuaj Mob Zoo (GCLP) rau kev kuaj mob raws li molecular siv hauv cov chaw kuaj mob, Hauv: Akyar I, tus kws kho. Cov spectra dav dav ntawm kev tswj xyuas zoo. Rijeka, Croatia: Intech; 2011: 29–52.
Lub sijhawm tshaj tawm: Lub Xya Hli-16-2020
