Mekhoa ea ho lemoha limolek'hule e na le bokhoni ba ho hlahisa palo e kholo ea asiti ea nucleic ka ho holisa lipalo tse fumanoang lisampoleng.Le hoja sena se le molemo bakeng sa ho nolofalletsa ho lemoha ka hloko, se boetse se hlahisa monyetla oa ho silafala ka ho hasana ha li-aerosol tsa amplification tikolohong ea laboratori.Ha ho etsoa liteko, ho ka etsoa mehato ea ho qoba tšilafalo ea li-reagents, lisebelisoa tsa laboratori le sebaka sa benche, kaha tšilafalo e joalo e ka hlahisa liphetho tse fosahetseng (kapa tse fosahetseng).

Ho thusa ho fokotsa menyetla ea tšoaetso, Boikoetliso bo Botle ba Laboratori bo lokela ho sebelisoa ka linako tsohle.Haholo-holo, mehato ea ho itšireletsa e lokela ho nkoa ka lintlha tse latelang:

1. Ho sebetsana le li-reagents
2. Tlhophiso ea sebaka sa mosebetsi le lisebelisoa
3. Sebelisa le ho hloekisa keletso bakeng sa sebaka se khethiloeng sa limolek'hule
4. Keletso e akaretsang ea baeloji ea limolek'hule
5. Litaolo tsa ka hare
6. Bukana ea libuka

1. Ho sebetsana le li-reagents

Ka bokhutšoane li-tubes tsa reagent tsa centrifuge pele li buloa ho qoba tlhahiso ea li-aerosol.Li-reagents tsa Aliquot ho qoba ho qhibiliha ka makhetlo a mangata le tšilafalo ea li-stocks tse kholo.Ngola ka ho hlaka, 'me u behe nako ka li-tubes tsohle tsa reagent le tsa reaction 'me u boloke lintlha tsa lotho le linomoro tsa batch tse sebelisoang litekong tsohle.Pipette li-reagents tsohle le lisampole u sebelisa malebela a sefe.Pele o reka, ho eletsoa ho netefatsa le moetsi hore lintlha tsa filthara li lumellana le mofuta oa pipette o lokelang ho sebelisoa.

2. Tlhophiso ea sebaka sa mosebetsi le lisebelisoa

Sebaka sa mosebetsi se lokela ho hlophisoa ho netefatsa hore ho phalla ha mosebetsi ho etsahala ka tsela e le 'ngoe, ho tloha libakeng tse hloekileng (pele ho PCR) ho ea libakeng tse litšila (post-PCR).Litlhokomelo tse latelang tse akaretsang li tla thusa ho fokotsa monyetla oa tšoaetso.E-ba le likamore tse arohaneng tse khethiloeng, kapa bonyane libaka tse arohaneng tsa 'mele, bakeng sa: ho lokisa motsoako oa mastermix, nucleic acid extract le DNA template, ho hōlisa le ho sebetsana le sehlahisoa sa amplified, le tlhahlobo ea lihlahisoa, mohlala gel electrophoresis.

Libakeng tse ling, ho thata ho ba le likamore tse 4 tse arohaneng.Khetho e ka khonehang empa e sa lakatseheng haholo ke ho etsa boitokisetso ba mastermix sebakeng sa polokelo, mohlala, laminar flow cabinet.Tabeng ea amplification ea PCR e entsoeng, ho lokisoa ha mastermix bakeng sa karabelo ea potoloho ea bobeli ho lokela ho lokisoa sebakeng se "hloekileng" bakeng sa ho lokisetsa mastermix, empa ente e nang le sehlahisoa sa mantlha sa PCR e lokela ho etsoa ka kamoreng ea amplification, 'me haeba ho khoneha. sebakeng se khethehileng sa polokelo (mohlala, laminar flow cabinet).

Kamore/sebaka se seng le se seng se hloka sete e arohaneng ea lipeipi tse ngotsoeng ka ho hlaka, malebela a sefa, li-tube racks, li-vortexes, li-centrifuges (haeba li le teng), lipene, li-reagents tsa generic lab, lijase tsa lab le mabokose a liatlana tse tla sala libakeng tsa tsona tsa mosebetsi.Matsoho a tlameha ho hlatsuoa 'me liatlana le lijase tsa lab li fetoloe ha li tsamaea lipakeng tsa libaka tse khethiloeng.Li-reagents le lisebelisoa ha lia lokela ho tlosoa sebakeng se litšila ho ea sebakeng se hloekileng.Haeba boemo bo feteletseng bo ka hlaha moo reajente kapa sengoathoana sa sesebelisoa se hlokang ho khutlisetsoa morao, se tlameha ho qala ka ho silafatsoa ka sodium hypochlorite ea 10%, ebe ho hlakoloa ka metsi a sa hloekang.

Hlokomela

Tharollo ea 10% ea sodium hypochlorite e tlameha ho etsoa e ncha letsatsi le letsatsi.Ha e sebelisoa bakeng sa ho tlosa tšilafalo, nako e fokolang ea ho kopana ea metsotso e 10 e lokela ho khomareloa.
Ntle le moo, lihlahisoa tse fumanehang khoebong tse netefalitsoeng e le li-decontaminants tse senyang DNA li ka sebelisoa haeba likhothaletso tsa polokeho ea lehae li sa lumelle tšebeliso ea sodium hypochlorite kapa sodium hypochlorite e sa loka bakeng sa ho silafatsa likarolo tsa tšepe tsa lisebelisoa.

Ha e le hantle, basebetsi ba lokela ho latela mekhoa e sa tšoaneng ea ho phalla ha mosebetsi 'me ba se ke ba tloha libakeng tse litšila (post-PCR) ho khutlela libakeng tse hloekileng (pele ho PCR) letsatsing lona leo.Leha ho le joalo, ho ka 'na ha e-ba le linako tseo sena se ke keng sa qojoa.Ha ketsahalo e joalo e hlaha, basebetsi ba tlameha ho hlokomela ho hlapa matsoho ka botlalo, ho fetola liatlana, ho sebelisa jase e khethiloeng ea lab le ho se hlahise thepa leha e le efe eo ba tla batla ho e ntša ka phaposing hape, joalo ka libuka tsa lab.Mehato e joalo ea taolo e lokela ho hatisoa thupelong ea basebetsi mabapi le mekhoa ea limolek'hule.

Ka mor'a tšebeliso, libaka tsa libenche li lokela ho hloekisoa ka 10% ea sodium hypochlorite (e lateloa ke metsi a hloekisitsoeng ho tlosa bleach e setseng), 70% ea ethanol, kapa ntho e nang le tšoaetso ea DNA e fumanehang khoebong.Ha e le hantle, mabone a ultra-violet (UV) a lokela ho kengoa ho thusa ho tlosa tšilafalo ka mahlaseli.Leha ho le joalo, tšebeliso ea mabone a UV e lokela ho etsoa feela libakeng tseo ho sebetsoang ho tsona tse koetsoeng, mohlala, lik'habote tsa tšireletso, e le ho fokotsa mahlaseli a mahlaseli a kotsi a basebetsi ba laboratori.Ka kopo, latela litaelo tsa moetsi bakeng sa tlhokomelo ea lebone la UV, moea le ho hloekisa ho netefatsa hore mabone a lula a sebetsa.

Haeba o sebelisa 70% ea ethanol sebakeng sa sodium hypochlorite, ho tla hlokahala mabone a UV ho phethela ts'oaetso.
U se ke ua hloekisa vortex le centrifuge ka sodium hypochlorite;ho e-na le hoo, hlakola ka 70% ea ethanol 'me u pepese khanya ea UV, kapa u sebelise DNA-e senyang decontaminant ea khoebo.Bakeng sa ho qhalana, ikopanye le moetsi bakeng sa keletso e eketsehileng ea ho hloekisa.Haeba litaelo tsa moetsi li e lumella, li-pipette li lokela ho hloekisoa ka tloaelo ka autoclave.Haeba li-pipette li ke ke tsa etsoa autoclaved, ho lokela ho lekane ho li hloekisa ka 10% sodium hypochlorite (e lateloa ke ho hlakola ka ho feletseng ka metsi a sa hloekang) kapa ka ntho e senyang DNA ea khoebo e lateloang ke ho pepeseha ha UV.

Ho hloekisa ka tekanyo e phahameng ea sodium hypochlorite qetellong ho ka 'na ha senya lipolasetiki tsa pipette le tšepe haeba li etsoa kamehla;hlahloba litlhahiso tse tsoang ho moetsi pele.Lisebelisoa tsohle li hloka ho lekanyetsoa khafetsa ho latela kemiso e khothaletsoang ke moetsi.Motho ea khethiloeng o lokela ho ikarabella ho netefatsa hore kemiso ea litekanyo ea lateloa, lintlha tse qaqileng lia hlokomeloa, 'me li-leibole tsa litšebeletso li behiloe ka ho hlaka holim'a thepa.

3. Sebelisa le ho hloekisa keletso bakeng sa sebaka se khethiloeng sa limolek'hule

Pre-PCR: Reagent aliquoting / mastermix preparation: Ena e lokela ho ba sebaka se hloekileng ka ho fetisisa ho feta libaka tsohle tse sebelisetsoang ho etsa liteko tsa limolek'hule 'me e lokela ho ba khabinete e khethiloeng ea laminar e nang le lebone la UV.lisampole, nucleic acid e nkiloeng le lihlahisoa tsa PCR tse holisitsoeng ha lia lokela ho sebetsoa sebakeng sena.Li-reagents tsa amplification li lokela ho bolokoa ka sehatsetsing (kapa sehatsetsing, joalo ka likhothaletso tsa moetsi) sebakeng se le seng se khethiloeng, hantle haufi le khabinete ea phallo ea laminar kapa sebaka sa pele ho PCR.Liatlana li lokela ho fetoloa nako le nako ha li kena sebakeng sa pele ho PCR kapa khabinete ea phallo ea laminar.

Sebaka sa pele ho PCR kapa khabinete ea phallo ea laminar e lokela ho hloekisoa pele le ka mor'a tšebeliso ka tsela e latelang: Hlakola lintho tsohle tse ka har'a khabinete, mohlala, li-pipette, li-tip box, vortex, centrifuge, li-tube racks, lipene, joalo-joalo ka 70% ea ethanol kapa DNA e senyang decontaminant ea khoebo, 'me e lumelle ho omella.Tabeng ea sebaka sa ts'ebetso se koetsoeng, mohlala, khabinete ea phallo ea laminar, pepesetsa lebone la UV bakeng sa metsotso e 30.

Hlokomela

Se ke oa pepesa li-reagents maboneng a UV;li ise feela ka hara khabinete ha e se e hloekile.Haeba o etsa PCR ea reverse, ho ka thusa hape ho hlakola bokaholimo le lisebelisoa ka tharollo e senyang RNase ha u kopana.Sena se ka thusa ho qoba liphetho tse fosahetseng tse tsoang ho ho senyeha ha enzyme ea RNA.Ka mor'a ho hloekisoa le pele u lokisetsa mastermix, liatlana li lokela ho fetoloa hape, ebe khabinete e se e loketse ho sebelisoa.

Pre-PCR: Ho ntšoa ha Nucleic acid / template ea tlatsetso:

Nucleic acid e tlameha ho ntšoa le ho sebetsoa sebakeng sa bobeli se khethiloeng, ho sebelisoa sete e arohaneng ea li-pipette, malebela a sefang, li-tube racks, liatlana tse foreshe, lijase tsa lab le lisebelisoa tse ling. mastermix tubes kapa plates.E le ho qoba ho silafala ha lisampole tse nkiloeng tsa nucleic acid tse ntseng li hlahlojoa, ho kgothaletswa hore o fetole liatlana pele o sebetsana le taolo kapa litekanyetso tse ntle le ho sebelisa sete e fapaneng ea pipette.Li-reagents tsa PCR le lihlahisoa tse matlafalitsoeng ha lia lokela ho tšeloa ka liphaephe sebakeng sena.Mehlala e lokela ho bolokoa ka lihatsetsing tse khethiloeng kapa lihatsetsi sebakeng se le seng.Sebaka sa ho sebetsa sa mohlala se lokela ho hloekisoa ka tsela e ts'oanang le sebaka sa mastermix.

Post-PCR: Ho holisa le ho sebetsana le sehlahisoa se holisitsoeng

Sebaka sena se khethiloeng ke sa lits'ebetso tsa kamora ho holisa 'me se lokela ho arohana le libaka tsa pele ho PCR.Hangata e na le li-thermocyclers le li-platform tsa nako ea sebele, 'me ka nepo e lokela ho ba le khabinete ea phallo ea laminar bakeng sa ho eketsa sehlahisoa sa 1 PCR ho karabelo e potolohileng 2, haeba PCR e behiloeng e ntse e etsoa.Li-reagents tsa PCR le nucleic acid tse nkiloeng ha lia lokela ho sebetsoa sebakeng sena kaha kotsi ea tšoaetso e phahame.Sebaka sena se lokela ho ba le sete e arohaneng ea liatlana, lijase tsa lab, li-plate le li-tube racks, li-pipette, malebela a sefa, meqomo le lisebelisoa tse ling.Li-tubes li tlameha ho kenngoa bohareng pele li buloa.Sebaka sa ho sebetsa sa mohlala se lokela ho hloekisoa ka tsela e ts'oanang le sebaka sa mastermix.

Post-PCR: Tlhahlobo ea lihlahisoa

Kamore ena ke ea lisebelisoa tsa ho lemoha lihlahisoa, mohlala, litanka tsa gel electrophoresis, lipakete tsa motlakase, transilluminator ea UV le sistimi ea litokomane tsa gel.Sebaka sena se lokela ho ba le lihlopha tse arohaneng tsa liatlana, lijase tsa lab, li-plate le li-tube racks, li-pipette, malebela a sefa, meqomo le lisebelisoa tse ling.Ha ho li-reagents tse ling tse ka tlisoang sebakeng sena, ntle le dae ea ho kenya, lets'oao la molek'hule le gel ea agarose, le likarolo tsa buffer.Sebaka sa ho sebetsa sa mohlala se lokela ho hloekisoa ka tsela e ts'oanang le sebaka sa mastermix.

Molaetsa oa bohlokoa

Ka mokhoa o loketseng, likamore tsa pele ho PCR ha lia lokela ho kenngoa ka letsatsi le le leng haeba mosebetsi o se o entsoe ka likamoreng tsa post-PCR.Haeba sena se ke ke sa qojoa ka ho felletseng, etsa bonnete ba hore matsoho a hlatsuoa hantle pele le hore lijase tsa lab tse khethehileng li apesoa ka likamoreng.Libuka tsa Lab le lipampiri ha lia lokela ho isoa likamoreng tsa pele ho PCR haeba li sebelisitsoe likamoreng tsa post-PCR;ha ho hlokahala, nka likopi tse hatisitsoeng tsa liprothokholo/ sampole ea li-ID, jj.

4. Keletso e akaretsang ea baeloji ea limolek'hule

Sebelisa liatlana tse se nang phofo ho qoba ho thibela liteko.Mokhoa o nepahetseng oa ho kenya lipeipi ke oa bohlokoa ho fokotsa tšilafalo.Lipeipi tse fosahetseng li ka fella ka ho fafatsa ha metsi le ho hlahisa li-aerosols.Mokhoa o motle oa ho kenya liphaephe tse nepahetseng o ka fumanoa lihokelong tse latelang: Tataiso ea Gilson ho lipeipi, livideo tsa theknoloji ea Anachem, Li-tubes tsa Centrifuge pele li bula, 'me u li bule ka hloko ho qoba ho phatloha.Koala li-tubes hang ka mor'a hore u sebelise ho qoba ho kenngoa ha litšila.

Ha o etsa karabelo e mengata, lokisa motsoako o le mong o nang le li-reagents tse tloaelehileng (mohlala, metsi, dNTPs, buffer, primers le enzyme) ho fokotsa palo ea phetisetso ea reagent le ho fokotsa ts'okelo ea tšilafalo.Ho kgothaletswa ho theha mastermix holim'a leqhoa kapa thibela e batang.Tšebeliso ea enzyme ea Hot Start e ka thusa ho fokotsa tlhahiso ea lihlahisoa tse sa tobang.Sireletsa li-reagents tse nang le lisebelisoa tsa fluorescent ho tloha leseling ho qoba ho senyeha.

5. Litaolo tsa ka hare

Kenyelletsa litaolo tse nang le litšobotsi tse ntle, tse tiisitsoeng tse ntle le tse mpe, hammoho le taolo e se nang thempleite mekhoeng eohle ea maikutlo, le mokhoa o nang le lintlha tse ngata bakeng sa maikutlo a bongata.Taolo e ntle ha ea lokela ho ba matla hoo e ka bakang kotsi ea tšoaetso.Kenyelletsa li-control tse ntle le tse mpe ha u etsa nucleic acid extract.

Ho khothalletsoa hore ho behoe litaelo tse hlakileng sebakeng se seng le se seng e le hore basebelisi ba tsebe melao ea boitšoaro.Li-lab tsa tlhahlobo tse fumanang maemo a tlase haholo a DNA kapa RNA lisampoleng tsa kliniki li kanna tsa batla ho amohela tekanyo e eketsehileng ea ts'ireletso ea ho ba le litsamaiso tse arohaneng tsa moea tse nang le khatello e ntle ea moea likamoreng tsa pele ho PCR le khatello e seng mpe ea moea likamoreng tsa kamora PCR.

Qetellong, ho etsa moralo oa boleng ba boleng (QA) hoa thusa.Morero o joalo o lokela ho kenyelletsa lethathamo la li-reagent master stocks le li-work stocks, melao ea ho boloka lisebelisoa le li-reagents, tlaleho ea liphetho tsa taolo, mananeo a koetliso ea basebetsi, mekhoa ea ho rarolla mathata, le mehato ea ho lokisa ha ho hlokahala.

6. Bukana ea libuka

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