Njira zodziwira mamolekyulu zimatha kupanga kuchuluka kwakukulu kwa nucleic acid kudzera pakukulitsa kuchuluka kwazomwe zimapezeka mu zitsanzo.Ngakhale izi ndizopindulitsa pakupangitsa kuti zizindikiridwe movutikira, zimabweretsanso kuthekera kwa kuipitsidwa mwa kufalikira kwa ma aerosols okulitsa mumalo a labotale.Poyesa kuyesa, njira zitha kuchitidwa pofuna kupewa kuipitsidwa kwa ma reagents, zida za labotale ndi malo a benchi, chifukwa kuipitsidwa kumeneku kungayambitse zotsatira zabodza (kapena zabodza).

Kuti muchepetse kuipitsidwa, Njira Yabwino ya Laboratory iyenera kuchitidwa nthawi zonse.Makamaka, kusamala kuyenera kutsatiridwa pazifukwa izi:

1. Kusamalira ma reagents
2. Kukonzekera kwa malo ogwirira ntchito ndi zipangizo
3. Kugwiritsa ntchito ndi kuyeretsa upangiri wa malo omwe asankhidwa
4. Malangizo a zamoyo zonse za maselo
5. Kuwongolera mkati
6. Zolemba

1. Kusamalira ma reagents

Mwachidule machubu a centrifuge reagent musanatsegule kupewa kutulutsa kwa aerosol.Ma reagents a Aliquot kuti apewe kuzizira kochulukirapo komanso kuipitsidwa kwa masheya apamwamba.Lembetsani momveka bwino ndikuwonetsa machubu onse a reagent ndi reaction ndikusunga zipika za reagent lot ndi manambala a batch omwe amagwiritsidwa ntchito pazoyeserera zonse.Pipette ma reagents onse ndi zitsanzo pogwiritsa ntchito nsonga zosefera.Musanayambe kugula, ndi bwino kutsimikizira ndi wopanga kuti nsonga za fyuluta zimagwirizana ndi mtundu wa pipette kuti ugwiritsidwe ntchito.

2. Kukonzekera kwa malo ogwirira ntchito ndi zipangizo

Malo ogwirira ntchito ayenera kukonzedwa kuti awonetsetse kuti kuyenda kwa ntchito kumachitika kumbali imodzi, kuchokera kumalo oyera (pre-PCR) kupita kumalo odetsedwa (post-PCR).Njira zodzitetezera zotsatirazi zithandiza kuchepetsa mwayi wotenga kachilomboka.Khalani ndi zipinda zosiyana, kapena m'malo otalikirana, monga: kukonzekera kwa mastermix, nucleic acid m'zigawo ndi kuwonjezera pa template ya DNA, kukulitsa ndi kusamalira zinthu zokulitsa, ndi kusanthula kwazinthu, mwachitsanzo gel electrophoresis.

M'malo ena, kukhala ndi zipinda zinayi zosiyana kumakhala kovuta.Njira yotheka koma yocheperako ndiyo kukonza zosakaniza m'malo osungiramo zinthu, monga laminar flow cabinet.Pankhani ya kukulitsa kwa PCR yokhala ndi zisa, kukonzekera kwa mastermix kwa gawo lachiwiri lozungulira kuyenera kukonzedwa m'malo 'oyera' pokonzekera mastermix, koma kulowetsedwa ndi chinthu choyambirira cha PCR kuyenera kuchitidwa mu chipinda chokulitsa, ndipo ngati n'kotheka. m'malo osungiramo zinthu (monga laminar flow cabinet).

Chipinda chilichonse/malo amafunikira ma pipette olembedwa momveka bwino, nsonga zosefera, zoyika machubu, ma vortexes, ma centrifuges (ngati kuli koyenera), zolembera, ma reagents amtundu wa lab, malaya a labotale ndi mabokosi a magolovesi omwe azikhala pamalo awo antchito.Manja ayenera kusambitsidwa ndi kusinthidwa magolovesi ndi malaya a labu pamene akuyenda pakati pa malo omwe asankhidwa.Ma reagents ndi zida siziyenera kusunthidwa kuchokera pamalo akuda kupita kumalo oyera.Pakachitika vuto lalikulu pomwe chowongolera kapena chida chikufunika kubwezeredwa chammbuyo, chiyenera kuchotsedwa ndi 10% sodium hypochlorite, kenako ndikupukuta ndi madzi osabala.

Zindikirani

10% sodium hypochlorite solution iyenera kupangidwa mwatsopano tsiku lililonse.Mukagwiritsidwa ntchito pochotsa matenda, nthawi yolumikizana ndi mphindi 10 iyenera kutsatiridwa.
Kapenanso, zinthu zomwe zimapezeka pamalonda zomwe zimatsimikiziridwa ngati zowononga DNA zowononga pamwamba zitha kugwiritsidwa ntchito ngati malingaliro achitetezo akumaloko salola kugwiritsa ntchito sodium hypochlorite kapena sodium hypochlorite si yoyenera kuwononga zida zachitsulo.

Moyenera, ogwira ntchito akuyenera kutsatira njira zoyendetsera ntchito zomwe sizikuyenda bwino komanso kuti asachoke kumalo odetsedwa (post-PCR) kubwerera kumadera oyera (pre-PCR) tsiku lomwelo.Komabe, pangakhale nthawi zina pamene izi sizingatheke.Izi zikachitika, ogwira ntchito ayenera kusamala kusamba m'manja, kusintha magolovesi, kugwiritsa ntchito jasi la labu lomwe asankhidwa komanso kuti asatchulenso zida zilizonse zomwe angafune kutulutsanso m'chipindamo, monga mabuku a labu.Njira zowongolera zotere ziyenera kugogomezera pakuphunzitsidwa kwa ogwira ntchito pa njira zama cell.

Mukagwiritsidwa ntchito, malo a benchi ayenera kutsukidwa ndi 10% sodium hypochlorite (motsatira madzi osabala kuti achotse bleach yotsalira), 70% ethanol, kapena mankhwala ovomerezeka omwe amawononga DNA.Moyenera, nyali za ultraviolet (UV) ziyenera kuikidwa kuti zithetse kuipitsidwa ndi kuwala.Komabe, kugwiritsa ntchito nyale za UV kuyenera kungokhala pamalo otsekedwa ogwirira ntchito, mwachitsanzo makabati achitetezo, kuti achepetse kukhudzidwa kwa ogwira ntchito mu labotale.Chonde tsatirani malangizo omwe amapanga pakusamalira nyale za UV, mpweya wabwino ndi kuyeretsa kuti mutsimikizire kuti nyali zikugwirabe ntchito.

Ngati mugwiritsa ntchito 70% ethanol m'malo mwa sodium hypochlorite, kuyatsa ndi kuwala kwa UV kudzafunika kuti mutsirize kuwononga.
Osayeretsa vortex ndi centrifuge ndi sodium hypochlorite;m'malo mwake, pukutani ndi 70% ethanol ndikuwonetsa kuwala kwa UV, kapena gwiritsani ntchito DNA-kuwononga decontaminant.Kuti muthe kutayikira, funsani kwa wopanga malangizo ena oyeretsa.Ngati malangizo opanga amalola, ma pipette ayenera kutsukidwa nthawi zonse ndi autoclave.Ngati ma pipette sangadzipangire okha, akuyenera kuwatsuka ndi 10% sodium hypochlorite (motsatira kupukuta mozama ndi madzi osabala) kapena ndi mankhwala owononga DNA-owononga otsatiridwa ndi kukhudzana ndi UV.

Kuyeretsa ndi sodium hypochlorite yapamwamba kwambiri kumatha kuwononga mapulasitiki a pipette ndi zitsulo ngati kuchitidwa pafupipafupi;fufuzani zoyamikira kuchokera kwa wopanga kaye.Zida zonse zimayenera kuyesedwa pafupipafupi malinga ndi ndondomeko yomwe wopanga amapangira.Munthu wosankhidwa ayenera kukhala ndi udindo wowonetsetsa kuti ndondomeko yowerengera ikutsatiridwa, zipika zatsatanetsatane zikusungidwa, ndipo zilembo zautumiki zikuwonetsedwa bwino pazida.

3. Kugwiritsa ntchito ndi kuyeretsa upangiri wa malo omwe asankhidwa

Pre-PCR: Reagent aliquoting / mastermix kukonzekera: Izi ziyenera kukhala zoyera kwambiri kuposa malo onse omwe amagwiritsidwa ntchito pokonzekera kuyesa kwa maselo ndipo ziyenera kukhala kabati yopangidwa ndi laminar yomwe ili ndi kuwala kwa UV.Zitsanzo, zotulutsidwa za nucleic acid ndi zinthu zokulitsa za PCR siziyenera kugwiridwa m'derali.Ma reagents okulitsa ayenera kusungidwa mufiriji (kapena mufiriji, malinga ndi malingaliro a wopanga) m'malo omwe asankhidwa, pafupi ndi kabati yotulutsa laminar kapena dera la PCR.Magolovesi ayenera kusinthidwa nthawi iliyonse mukalowa m'dera la pre-PCR kapena laminar flow cabinet.

Pre-PCR dera kapena laminar flow kabati ayenera kutsukidwa pamaso ndi pambuyo ntchito motere: Pukutani pansi zinthu zonse mu nduna, mwachitsanzo pipettes, nsonga mabokosi, vortex, centrifuge, chubu poyimitsa, zolembera, etc. ndi 70% Mowa kapena a malonda DNA-kuwononga decontaminant, ndi kulola kuti ziume.Pamalo otsekedwa ogwirira ntchito, mwachitsanzo, kabati yoyendera laminar, iwonetseni chophimbacho ku kuwala kwa UV kwa mphindi 30.

Zindikirani

Osawonetsa ma reagents ku kuwala kwa UV;kungowasuntha iwo mu kabati kamodzi ali woyera.Ngati mukupanga reverse transcription PCR, zingakhale zothandizanso kupukuta pansi ndi zida ndi yankho lomwe limaphwanya RNases mukakumana.Izi zingathandize kupewa zotsatira zabodza kuchokera ku kuwonongeka kwa ma enzyme a RNA.Pambuyo powonongeka komanso musanakonzekere mastermix, magolovesi ayenera kusinthidwa kamodzinso, ndiyeno kabati ndi yokonzeka kugwiritsa ntchito.

Pre-PCR: Kutulutsa kwa Nucleic acid / template yowonjezera:

Nucleic acid iyenera kuchotsedwa ndikusamalidwa pamalo achiwiri osankhidwa, pogwiritsa ntchito ma pipette, nsonga zosefera, ma chubu, magolovesi atsopano, malaya a labu ndi zida zina. mastermix machubu kapena mbale.Pofuna kupewa kuipitsidwa kwa zitsanzo za nucleic acid zomwe zikuwunikidwa, tikulimbikitsidwa kuti musinthe magolovesi musanagwiritse ntchito zowongolera kapena miyezo yabwino ndikugwiritsa ntchito ma pipette.Ma reagents a PCR ndi zinthu zokulitsa siziyenera kuponyedwa m'derali.Zitsanzo ziyenera kusungidwa mufiriji kapena mufiriji pamalo omwewo.Chitsanzo chogwirira ntchito chiyenera kutsukidwa mofanana ndi malo a mastermix.

Post-PCR: Kukulitsa ndi kasamalidwe ka mankhwala okulitsa

Malo osankhidwawa ndi a njira zokulirapo ndipo ayenera kukhala osiyana ndi ma PCR asanakhalepo.Nthawi zambiri imakhala ndi ma thermocyclers ndi nsanja zanthawi yeniyeni, ndipo iyenera kukhala ndi kabati yotulutsa laminar kuti iwonjezere mankhwala ozungulira 1 PCR pazozungulira 2, ngati PCR yosungidwa ikuchitika.PCR reagents ndi nucleic acid yotengedwa sayenera kugwiridwa m'derali chifukwa chiopsezo choipitsidwa ndi chachikulu.Derali liyenera kukhala ndi magolovesi, malaya a labu, mbale ndi ma chubu, ma pipette, nsonga zosefera, nkhokwe ndi zida zina.Machubu ayenera kukhala centrifuged asanatsegule.Chitsanzo chogwirira ntchito chiyenera kutsukidwa mofanana ndi malo a mastermix.

Post-PCR: Kusanthula kwazinthu

Chipindachi ndi cha zida zozindikirira zinthu, mwachitsanzo matanki a gel electrophoresis, mapaketi amagetsi, ma transilluminator a UV ndi makina osindikizira a gel.Derali liyenera kukhala ndi magulu osiyana a magolovesi, malaya a labu, mbale ndi ma chubu, ma pipette, nsonga zosefera, nkhokwe ndi zida zina.Palibe ma reagents ena omwe angabweretsedwe mderali, kupatula utoto wodzaza, cholembera ma molekyulu ndi gel agarose, ndi zida za buffer.Chitsanzo chogwirira ntchito chiyenera kutsukidwa mofanana ndi malo a mastermix.

Mfundo yofunika

Moyenera, zipinda za pre-PCR siziyenera kulowetsedwa tsiku lomwelo ngati ntchito yachitika kale m'zipinda za post-PCR.Ngati izi sizingalephereke, onetsetsani kuti m'manja mwasambitsidwa bwino komanso kuti zovala za labu zivale m'zipinda.Mabuku a labu ndi mapepala sayenera kutengedwera m'zipinda za Pre-PCR ngati zakhala zikugwiritsidwa ntchito m'zipinda za post-PCR;ngati kuli kofunikira, tengani zobwereza za ma protocol/ma ID a zitsanzo, ndi zina.

4. Malangizo a zamoyo zonse za maselo

Gwiritsani ntchito magolovesi opanda ufa kuti mupewe kulepheretsa kuyesa.Njira yoyenera yopangira mapaipi ndiyofunikira kwambiri kuti muchepetse kuipitsidwa.Kupaka mapaipi kolakwika kungayambitse kuwondana popereka zakumwa komanso kupanga ma aerosols.Kuchita bwino kwa mapaipi olondola kungapezeke pa maulalo otsatirawa: Gilson kalozera wa mapaipi, makanema aukadaulo a Anachem, machubu a Centrifuge musanatsegule, ndipo atseguleni mosamala kuti asaphulike.Tsekani machubu mukangogwiritsa ntchito kuti mupewe kuyambitsa zowononga.

Mukamachita zinthu zingapo, konzekerani mastermix imodzi yokhala ndi ma reagents wamba (monga madzi, dNTPs, buffer, primers ndi enzyme) kuti muchepetse kuchuluka kwa kusamutsidwa kwa reagent ndikuchepetsa kuwopsa kwa kuipitsidwa.Ndikofunikira kukhazikitsa mastermix pa ayezi kapena chipika chozizira.Kugwiritsa ntchito enzyme ya Hot Start kungathandize kuchepetsa kupanga zinthu zomwe sizikudziwika.Tetezani ma reagents okhala ndi ma probe a fulorosenti ku kuwala kuti mupewe kuwonongeka.

5. Kuwongolera mkati

Phatikizani maulamuliro odziwika bwino, otsimikiziridwa abwino ndi oyipa, limodzi ndi kuwongolera kopanda template pamachitidwe onse, ndi njira yotsatiridwa ndi mfundo zambiri zamachitidwe kachulukidwe.Kuwongolera kwabwino sikuyenera kukhala kolimba kwambiri kotero kuti kumabweretsa chiopsezo choyipitsidwa.Phatikizanipo zowongolera zabwino ndi zoyipa pakuchotsa nucleic acid.

Ndibwino kuti malangizo omveka bwino alembedwe m'madera onse kuti ogwiritsa ntchito adziwe malamulo a khalidwe.Ma labu ozindikira omwe amazindikira milingo yotsika kwambiri ya DNA kapena RNA m'zitsanzo zachipatala angafunike kutengera njira zina zachitetezo zokhala ndi makina osiyana siyana okhala ndi mpweya wabwino pang'ono m'zipinda za PCR asanakhale ndi mpweya woipa pang'ono m'zipinda za PCR.

Pomaliza, kupanga dongosolo lotsimikizira zamtundu (QA) ndikofunikira.Dongosolo lotereli liyenera kukhala ndi mndandanda wa masitoko a rejenti ndi masheya ogwirira ntchito, malamulo osungira zida ndi zopangira, lipoti la zotsatira zowongolera, mapologalamu ophunzitsira ogwira ntchito, ma aligorivimu othetsera mavuto, ndi zowongolera pakafunika.

6. Zolemba

Aslan A, Kinzelman J, Dreelin E, Anan'eva T, Lavander J. Mutu 3: Kukhazikitsa labotale ya qPCR.Chikalata chowongolera poyesa madzi osangalatsa pogwiritsa ntchito USEPA qPCR njira 1611. Lansing- Michigan State University.

Public Health England, NHS.Miyezo yaku UK yakufufuza kwa Microbiology: Kuchita Zabwino Kwa Laborator mukamayesa kuyesa kwa ma molekyulu).Upangiri Wabwino.2013;4(4):1–15.

Mifflin T. Kukhazikitsa labotale ya PCR.Cold Spring Harb Protoc.2007; 7.

Schroeder S.Hamburg: Eppendorf;2013.

Viana RV, Wallis CL.Good Clinical Laboratory Practice (GCLP) pamayeso otengera mamolekyulu omwe amagwiritsidwa ntchito m'ma laboratories ozindikira matenda, Mu: Akyar I, mkonzi.Kuwongolera kwamtundu wambiri.Rijeka, Croatia: Intech;2011: 29-52.