Njira zopezera mamolekyulu zimatha kupanga nucleic acid yambiri kudzera mu kukulitsa kuchuluka kwa ma trace omwe amapezeka mu zitsanzo. Ngakhale izi ndizothandiza pothandiza kuzindikira mwanzeru, zimayambitsanso kuthekera kwa kuipitsidwa kudzera mu kufalikira kwa ma aerosols okulitsa m'malo ochitira kafukufuku. Pochita zoyeserera, njira zitha kuchitidwa kuti mupewe kuipitsidwa kwa ma reagents, zida za labotale ndi malo osungiramo zinthu, chifukwa kuipitsidwa koteroko kungayambitse zotsatira zabodza (kapena zabodza).
Pofuna kuchepetsa kuipitsidwa kwa zinthu, njira zabwino zogwiritsira ntchito ma laboratory ziyenera kutsatiridwa nthawi zonse. Makamaka, njira zodzitetezera ziyenera kutengedwa pankhani zotsatirazi:
1. Kusamalira ma reagents
2. Kukonza malo ogwirira ntchito ndi zida
3. Malangizo ogwiritsira ntchito ndi kuyeretsa malo osankhidwa a mamolekyulu
4. Upangiri wa sayansi ya zamoyo zamamolekyulu
5. Zowongolera zamkati
6. Mabuku Olembedwa
1. Kusamalira ma reagents
Pakapita kanthawi, thirani machubu a reagent a centrifuge musanatsegule kuti mupewe kupanga ma aerosol. Ikani ma reagent kuti mupewe kuzizira kwambiri komanso kuipitsidwa kwa masitolo akuluakulu. Lembani mayina ndi tsiku la machubu onse a reagent ndi reaction ndikusunga zolemba za reagent lot ndi manambala a batch omwe amagwiritsidwa ntchito muzoyesa zonse. Pipette ma reagent ndi zitsanzo pogwiritsa ntchito ma fyuluta. Musanagule, ndibwino kutsimikizira ndi wopanga kuti ma fyuluta akugwirizana ndi mtundu wa pipette yomwe ikugwiritsidwa ntchito.
2. Kukonza malo ogwirira ntchito ndi zida
Malo ogwirira ntchito ayenera kukonzedwa kuti atsimikizire kuti kuyenda kwa ntchito kumachitika mbali imodzi, kuchokera kumadera oyera (pre-PCR) kupita kumadera auve (post-PCR). Malangizo otsatirawa athandiza kuchepetsa mwayi wodetsedwa. Khalani ndi zipinda zosiyana, kapena osachepera madera osiyana, kuti: kukonzekera mastermix, kuchotsa nucleic acid ndi kuwonjezera template ya DNA, kukulitsa ndi kusamalira mankhwala owonjezera, ndi kusanthula mankhwala, mwachitsanzo gel electrophoresis.
Mu malo ena, kukhala ndi zipinda zinayi zosiyana n'kovuta. Njira yotheka koma yosafunikira ndikuchita kukonzekera kwa mastermix pamalo osungira, mwachitsanzo kabati yoyendera madzi ya laminar. Pankhani ya kukulitsa kwa PCR komwe kumayikidwa nested, kukonzekera kwa mastermix kwa reaction yachiwiri kuyenera kukonzedwa pamalo 'oyera' kuti akonzekere mastermix, koma kuyika mankhwala oyamba a PCR kuyenera kuchitikira m'chipinda chowonjezera madzi, ndipo ngati n'kotheka m'malo osungira madzi apadera (monga kabati yoyendera madzi ya laminar).
Chipinda/malo aliwonse amafunika mapaipi osiyana olembedwa bwino, ma fyuluta, ma tube racks, ma vortexes, ma centrifuge (ngati kuli koyenera), mapeni, ma generic lab reagents, ma lab coats ndi mabokosi a magolovesi omwe azikhala m'malo awo ogwirira ntchito. Manja ayenera kutsukidwa ndipo magolovesi ndi ma lab coats ayenera kusinthidwa posuntha pakati pa malo osankhidwa. Ma reagents ndi zida siziyenera kusunthidwa kuchokera pamalo odetsedwa kupita kumalo oyera. Ngati pakhala vuto lalikulu pamene reagent kapena chipangizocho chikufunika kusunthidwa kumbuyo, choyamba chiyenera kutsukidwa ndi 10% sodium hypochlorite, kenako kutsukidwa ndi madzi oyera.
Zindikirani
Mankhwala a 10% sodium hypochlorite ayenera kupangidwa mwatsopano tsiku lililonse. Mukagwiritsidwa ntchito pochotsa kuipitsidwa, nthawi yocheperako yokhudza mankhwalawa iyenera kutsatiridwa kwa mphindi 10.
Kapenanso, zinthu zomwe zimapezeka m'masitolo zomwe zatsimikiziridwa kuti ndi zotsukira pamwamba zomwe zimawononga DNA zingagwiritsidwe ntchito ngati malangizo achitetezo am'deralo salola kugwiritsa ntchito sodium hypochlorite kapena ngati sodium hypochlorite si yoyenera kuchotsa kuipitsidwa kwa ziwalo zachitsulo za zida.
Mwanjira yabwino, ogwira ntchito ayenera kutsatira mfundo ya kayendetsedwe ka ntchito kamodzi kokha ndipo asasiye malo odetsedwa (post-PCR) kubwerera kumalo oyera (pre-PCR) tsiku lomwelo. Komabe, pakhoza kukhala nthawi zina pamene izi sizingatheke. Ngati izi zitachitika, ogwira ntchito ayenera kusamala kuti asambe m'manja mokwanira, kusintha magolovesi, kugwiritsa ntchito chovala cholembedwa cha labu ndipo asabweretse zida zilizonse zomwe angafune kuchotsa m'chipindamo kachiwiri, monga mabuku a labu. Njira zowongolera zoterezi ziyenera kutsindika pophunzitsa ogwira ntchito njira zamamolekyu.
Mukamaliza kugwiritsa ntchito, malo ogwirira ntchito ayenera kutsukidwa ndi 10% sodium hypochlorite (kutsatiridwa ndi madzi osabala kuti achotse bleach yotsala), 70% ethanol, kapena chotsukira DNA chovomerezeka chomwe chikupezeka pamsika. Mwabwino, nyali za ultra-violet (UV) ziyenera kuyikidwa kuti zithetse kuipitsidwa ndi kuwala. Komabe, kugwiritsa ntchito nyali za UV kuyenera kungokhala m'malo otsekedwa ogwirira ntchito, mwachitsanzo makabati oteteza, kuti achepetse kuwonekera kwa UV kwa ogwira ntchito m'chipatala. Chonde tsatirani malangizo a wopanga pakusamalira nyali za UV, mpweya wabwino komanso kuyeretsa kuti nyali zipitirize kugwira ntchito bwino.
Ngati mugwiritsa ntchito 70% ethanol m'malo mwa sodium hypochlorite, kuyatsa ndi kuwala kwa UV kudzafunika kuti mumalize kuchotsa kuipitsidwa.
Musatsuke vortex ndi centrifuge ndi sodium hypochlorite; m'malo mwake, pukutani ndi 70% ethanol ndikuyika ku kuwala kwa UV, kapena gwiritsani ntchito chotsukira DNA chogulitsa. Ngati mwataya, funsani wopanga kuti akupatseni malangizo ena oyeretsa. Ngati malangizo a wopanga alola, ma pipettes ayenera kutsukidwa nthawi zonse ndi autoclave. Ngati ma pipettes sangathe kutsukidwa, ayenera kutsukidwa ndi 10% sodium hypochlorite (kutsatiridwa ndi kupukuta bwino ndi madzi osayera) kapena ndi chotsukira DNA chogulitsa chomwe chimawononga kutsatiridwa ndi UV.
Kuyeretsa ndi sodium hypochlorite yochuluka kumatha kuwononga mapulasitiki ndi zitsulo ngati kuchitidwa nthawi zonse; choyamba yang'anani malangizo ochokera kwa wopanga. Zipangizo zonse ziyenera kuyesedwa nthawi zonse malinga ndi ndondomeko yomwe wopanga amalangiza. Munthu wosankhidwa ayenera kuyang'anira kuonetsetsa kuti ndondomeko yowunikira ikutsatira, zolemba zambiri zikusungidwa, ndipo zilembo zautumiki zikulembedwa bwino pazida.
3. Malangizo ogwiritsira ntchito ndi kuyeretsa malo osankhidwa a mamolekyulu
Pre-PCR: Kukonzekera kwa Reagent / mastermix: Iyi iyenera kukhala malo oyera kwambiri omwe amagwiritsidwa ntchito pokonzekera zoyeserera zamamolekyu ndipo iyenera kukhala kabati yoyendera ya laminar yokhala ndi kuwala kwa UV. Zitsanzo, nucleic acid yochotsedwa ndi zinthu za PCR zokwezedwa siziyenera kusungidwa m'derali. Ma reagent owonjezera ayenera kusungidwa mufiriji (kapena firiji, malinga ndi malangizo a wopanga) m'malo omwewo, makamaka pafupi ndi kabati yoyendera ya laminar kapena dera la pre-PCR. Magolovesi ayenera kusinthidwa nthawi iliyonse mukalowa m'dera la pre-PCR kapena kabati yoyendera ya laminar.
Malo ogwiritsira ntchito PCR kapena kabati yoyendera madzi iyenera kutsukidwa musanagwiritse ntchito komanso mutagwiritsa ntchito motere: Pukutani zinthu zonse zomwe zili mu kabati, mwachitsanzo ma pipettes, mabokosi a tip, vortex, centrifuge, ma tube racks, ma pen, ndi zina zotero ndi 70% ethanol kapena chotsukira DNA chogulitsa, ndipo mulole kuti chiume. Ngati malo ogwirira ntchito atsekedwa, mwachitsanzo kabati yoyendera madzi, onetsani hood ku kuwala kwa UV kwa mphindi 30.
Zindikirani
Musamaike ma reagents ku kuwala kwa UV; ingowalowetsani mu kabati mukangoyera. Ngati mukuchita reverse transcription PCR, zingakhalenso zothandiza kupukuta pamwamba ndi zida ndi yankho lomwe limaphwanya ma RNases akakhudzana. Izi zingathandize kupewa zotsatira zabodza kuchokera ku kuwonongeka kwa enzyme ya RNA. Mukachotsa kuipitsidwa komanso musanakonze mastermix, magolovesi ayenera kusinthidwanso, kenako kabatiyo ikhale yokonzeka kugwiritsidwa ntchito.
Pre-PCR: Kuchotsa/kuwonjezera template ya Nucleic acid:
Nucleic acid iyenera kuchotsedwa ndikugwiridwa m'malo ena osankhidwa, pogwiritsa ntchito seti yosiyana ya ma pipettes, ma fyuluta, ma tube racks, magolovesi atsopano, ma lab coats ndi zida zina. Malo awa ndi owonjezeranso template, zowongolera ndi mizere yoyendera ku ma mastermix tubes kapena ma plates. Kuti mupewe kuipitsidwa kwa zitsanzo za nucleic acid zomwe zachotsedwa zomwe zikuwunikidwa, tikukulimbikitsani kusintha magolovesi musanayambe kugwiritsa ntchito zowongolera zabwino kapena miyezo ndikugwiritsa ntchito seti yosiyana ya ma pipettes. Ma PCR reagents ndi zinthu zowonjezera siziyenera kuyikidwa m'malo awa. Zitsanzo ziyenera kusungidwa m'mafiriji kapena ma freezers omwe adasankhidwa m'malo omwewo. Malo ogwirira ntchito oyeserera ayenera kutsukidwa mofanana ndi malo a mastermix.
Pambuyo pa PCR: Kukulitsa ndi kusamalira mankhwala owonjezera
Malo osankhidwa awa ndi a ntchito zotsatizana pambuyo pa kukulitsa ndipo ayenera kukhala osiyana ndi madera omwe asanayambe PCR. Nthawi zambiri amakhala ndi ma thermocycler ndi mapulatifomu a nthawi yeniyeni, ndipo ndibwino kukhala ndi kabati yoyendera madzi yowonjezerera chinthu chozungulira cha PCR 1 ku reaction yozungulira 2, ngati PCR yokhala ndi nested ikuchitidwa. Ma PCR reagents ndi nucleic acid yotulutsidwa sayenera kusamalidwa m'derali chifukwa chiopsezo cha kuipitsidwa ndi chachikulu. Malowa ayenera kukhala ndi magolovesi osiyana, ma lab coats, ma plate ndi ma tube racks, ma pipettes, ma filter tips, ma bin ndi zida zina. Ma chubu ayenera kuyikidwa pakati asanatsegulidwe. Malo ogwirira ntchito oyeserera ayenera kutsukidwa mofanana ndi malo a mastermix.
Pambuyo pa PCR: Kusanthula kwa malonda
Chipinda chino ndi cha zipangizo zodziwira zinthu, mwachitsanzo matanki a gel electrophoresis, ma power packs, UV transilluminator ndi makina olembera ma gel. Malo awa ayenera kukhala ndi magolovesi osiyana, ma lab coats, ma plate and tube racks, ma pipettes, ma filter tips, ma bin ndi zida zina. Palibe ma reagents ena omwe angabweretsedwe m'derali, kupatulapo loading dye, molecular marker ndi agarose gel, ndi zigawo zina za buffer. Malo ogwirira ntchito a chitsanzo ayenera kutsukidwa mofanana ndi malo a mastermix.
Chidziwitso chofunikira
Mwanjira yabwino, zipinda zogwiritsira ntchito PCR siziyenera kulowedwa tsiku lomwelo ngati ntchito yachitika kale m'zipinda zogwiritsira ntchito PCR. Ngati izi sizingatheke, onetsetsani kuti manja ayamba kutsukidwa bwino ndipo malaya enaake a labu amavalidwa m'zipindazo. Mabuku ndi mapepala a labu sayenera kutengedwa m'zipinda zogwiritsira ntchito PCR ngati adagwiritsidwa ntchito m'zipinda zogwiritsira ntchito PCR; ngati kuli kofunikira, tengani mapepala osindikizidwa kawiri a protocol/sample ID, ndi zina zotero.
4. Upangiri wa sayansi ya zamoyo zamamolekyulu
Gwiritsani ntchito magolovesi opanda ufa kuti mupewe kuletsa mayeso. Njira yoyenera yogwiritsira ntchito mapaipi ndiyofunika kwambiri pochepetsa kuipitsidwa. Kugwiritsira ntchito mapaipi molakwika kungayambitse kupopera madzi potulutsa madzi ndi kupanga ma aerosol. Njira yabwino yogwiritsira ntchito mapaipi moyenera ingapezeke pa maulalo otsatirawa: Gilson guide to pipetting, Anachem pipetting technique videos, Centrifuge tubes musanatsegule, ndipo muwatsegule mosamala kuti mupewe kupopera madzi. Tsekani machubu nthawi yomweyo mukatha kugwiritsa ntchito kuti mupewe kulowetsa zinthu zodetsa.
Mukachita zinthu zosiyanasiyana, konzani mastermix imodzi yokhala ndi ma reagents ofanana (monga madzi, dNTPs, buffer, primers ndi enzyme) kuti muchepetse kuchuluka kwa ma reagents omwe amasamutsidwa ndikuchepetsa chiopsezo cha kuipitsidwa. Ndikofunikira kukhazikitsa mastermix pa ayezi kapena pamalo ozizira. Kugwiritsa ntchito enzyme ya Hot Start kungathandize kuchepetsa kupanga zinthu zinazake. Tetezani ma reagents okhala ndi ma fluorescent probes ku kuwala kuti mupewe kuwonongeka.
5. Zowongolera zamkati
Phatikizani zowongolera zabwino ndi zoyipa zomwe zatsimikiziridwa bwino, pamodzi ndi kuwongolera kopanda template pazochita zonse, ndi mzere wotsatira wa ma point angapo a zochita zowerengera. Kuwongolera kwabwino sikuyenera kukhala kolimba kwambiri kotero kuti kungayambitse kuipitsidwa. Phatikizani zowongolera zabwino ndi zoyipa pakuchotsa nucleic acid.
Ndikofunikira kuti malangizo omveka bwino aikidwe m'magawo onsewa kuti ogwiritsa ntchito adziwe malamulo oyendetsera zinthu. Ma labu ofufuza omwe amapeza kuchuluka kochepa kwa DNA kapena RNA m'zitsanzo zachipatala angafune kugwiritsa ntchito njira yowonjezera yotetezera yokhala ndi makina osiyana oyendetsera mpweya okhala ndi mpweya wabwino pang'ono m'zipinda zoyeserera PCR ndi mpweya woipa pang'ono m'zipinda zoyeserera PCR.
Pomaliza, kupanga dongosolo lotsimikizira khalidwe (QA) n'kothandiza. Dongosolo lotereli liyenera kukhala ndi mndandanda wa masheya akuluakulu a reagent ndi masheya ogwira ntchito, malamulo osungira zida ndi ma reagent, malipoti a zotsatira zowongolera, mapulogalamu ophunzitsira antchito, ma algorithms othetsera mavuto, ndi njira zothanirana ndi vutoli pakafunika kutero.
6. Mabuku Olembedwa
Aslan A, Kinzelman J, Dreelin E, Anan'eva T, Lavander J. Mutu 3: Kukhazikitsa labotale ya qPCR. Chikalata chotsogolera poyesa madzi osangalatsa pogwiritsa ntchito njira ya USEPA qPCR 1611. Lansing- Michigan State University.
Public Health England, NHS. Miyezo ya UK yofufuza za microbiology: Machitidwe Abwino a Laboratory pochita mayeso a molecular amplification). Malangizo Abwino. 2013;4(4):1–15.
Mifflin T. Kukhazikitsa labotale ya PCR. Cold Spring Harb Protoc. 2007;7.
Schroeder S 2013. Kusamalira nthawi zonse ma centrifuge: kuyeretsa, kukonza ndi kupha tizilombo toyambitsa matenda ma centrifuge, ma rotor ndi ma adapter (Pepala Loyera Nambala 14). Hamburg: Eppendorf; 2013.
Viana RV, Wallis CL. Good Clinical Laboratory Practice (GCLP) ya mayeso ozikidwa pa molekyulu omwe amagwiritsidwa ntchito m'ma laboratories ozindikira matenda, Mu: Akyar I, mkonzi. Ma spectra ambiri owongolera khalidwe. Rijeka, Croatia: Intech; 2011: 29–52.
Nthawi yotumizira: Julayi-16-2020
