Izindlela zokuthola ama-molecule zinekhono lokukhiqiza inani elikhulu le-nucleic acid ngokukhulisa inani lokulandelela elitholakala kumasampula. Nakuba lokhu kuzuzisa ukuvumela ukutholwa okubucayi, futhi kwethula ithuba lokungcola ngokusabalalisa ama-aerosol okukhulisa endaweni yelabhorethri. Lapho kwenziwa izivivinyo, kungenziwa izinyathelo zokugwema ukungcola kwama-reagents, imishini yelabhorethri kanye nendawo yebhentshi, njengoba ukungcola okunjalo kungadala imiphumela emibi (noma emibi).
Ukuze kuncishiswe amathuba okungcoliswa, kufanele kusetshenziswe uMkhuba Omuhle Welebhu ngaso sonke isikhathi. Ngokukhethekile, kufanele kuthathwe izinyathelo zokuphepha maqondana namaphuzu alandelayo:
1. Ukuphatha ama-reagent
2. Ukuhlela indawo yokusebenza kanye nemishini
3. Iseluleko sokusetshenziswa nokuhlanza isikhala sama-molecule esikhethiwe
4. Iseluleko esijwayelekile sebhayoloji yama-molecule
5. Izilawuli zangaphakathi
6. Uhlu lwezincwadi
1. Ukuphatha ama-reagent
Faka amashubhu e-reagent e-centrifuge kafushane ngaphambi kokuvula ukuze ugweme ukukhiqizwa kwama-aerosol. Hlanganisa ama-reagent ukuze ugweme ukuncibilika okuningi kweqhwa kanye nokungcola kwamasheya amakhulu. Bhala ilebula ngokucacile futhi ubhale usuku kuwo wonke amashubhu e-reagent kanye ne-reaction bese ugcina izingodo zenani lama-reagent kanye nezinombolo ze-batch ezisetshenziswa kuzo zonke izivivinyo. Faka ama-reagent namasampula usebenzisa amathiphu okuhlunga. Ngaphambi kokuthenga, kungcono ukuqinisekisa nomkhiqizi ukuthi amathiphu okuhlunga afanelana nohlobo lwe-pipette ezosetshenziswa.
2. Ukuhlela indawo yokusebenza kanye nemishini
Indawo yokusebenza kufanele ihlelwe ukuqinisekisa ukuthi ukuhamba komsebenzi kwenzeka ohlangothini olulodwa, kusukela ezindaweni ezihlanzekile (pre-PCR) kuya ezindaweni ezingcolile (post-PCR). Izinyathelo ezilandelayo zokuphepha ezijwayelekile zizosiza ukunciphisa amathuba okungcola. Yiba namakamelo ahlukene aqokiwe, noma okungenani ezindaweni ezihlukene ngokomzimba, ukuze: ukulungiswa kwe-mastermix, ukukhishwa kwe-nucleic acid kanye nokwengezwa kwethempulethi ye-DNA, ukwandiswa nokuphathwa komkhiqizo okhulisiwe, kanye nokuhlaziywa komkhiqizo, isib. i-gel electrophoresis.
Kwezinye izimo, ukuba namakamelo amane ahlukene kunzima. Inketho engenzeka kodwa engafiseleki kakhulu ukwenza ukulungiswa kwe-mastermix endaweni yokuvimba, isb. ikhabhinethi yokugeleza ye-laminar. Uma kwenzeka ukukhulisa i-PCR okufakwe esidlekeni, ukulungiswa kwe-mastermix yokusabela kwesibili kufanele kulungiselelwe endaweni 'ehlanzekile' yokulungiselela i-mastermix, kodwa ukugonywa ngomkhiqizo we-PCR oyinhloko kufanele kwenziwe egumbini lokukhulisa, futhi uma kungenzeka endaweni yokuvimba enikezelwe (isb. ikhabhinethi yokugeleza ye-laminar).
Igumbi/indawo ngayinye idinga isethi ehlukile yamapayipi abhalwe ngokucacile, amathiphu okuhlunga, ama-tube racks, ama-vortexes, ama-centrifuge (uma kufanele), amapeni, ama-reagents elebhu ejwayelekile, amajazi elebhu kanye namabhokisi amagilavu azosala ezindaweni zawo zokusebenza. Izandla kumele zigezwe futhi amagilavu kanye namajazi elebhu ashintshwe lapho kuhanjwa phakathi kwezindawo ezibekiwe. Ama-reagents kanye nemishini akufanele kususwe endaweni engcolile kuye endaweni ehlanzekile. Uma kuvela isimo esibi kakhulu lapho i-reagent noma ingxenye yemishini idinga ukuhlehliswa emuva, kumele kuqala ihlanzwe nge-10% sodium hypochlorite, kulandelwe ukusula ngamanzi ahlanzekile.
Inothi
Isixazululo se-sodium hypochlorite esingu-10% kumele senziwe sisha nsuku zonke. Uma sisetshenziselwa ukuhlanza amagciwane, kufanele kulandelwe isikhathi esincane sokuxhumana semizuzu eyi-10.
Ngaphandle kwalokho, imikhiqizo etholakala kwezentengiselwano eqinisekiswe njengezinto zokususa ukungcola ebusweni ezibhubhisa i-DNA ingasetshenziswa uma izincomo zokuphepha zendawo zingavumeli ukusetshenziswa kwe-sodium hypochlorite noma uma i-sodium hypochlorite ingafaneleki ukuhlanza izingxenye zensimbi zemishini.
Okungcono kakhulu, abasebenzi kufanele balandele umthetho wokuhamba komsebenzi oqondiswe ohlangothini olulodwa futhi bangasuki ezindaweni ezingcolile (ngemuva kwe-PCR) babuyele ezindaweni ezihlanzekile (ngaphambi kwe-PCR) ngosuku olufanayo. Kodwa-ke, kungase kube nezikhathi lapho lokhu kungenakugwenywa. Uma kuvela lesi simo, abasebenzi kumele baqaphele ukugeza izandla kahle, bashintshe amagilavu, basebenzise ijazi lelebhu elibekiwe futhi bangafaki noma yimiphi imishini abazofuna ukuyikhipha ekamelweni futhi, njengezincwadi zelebhu. Izinyathelo ezinjalo zokulawula kufanele zigcizelelwe ekuqeqeshweni kwabasebenzi ngezindlela zama-molecule.
Ngemva kokusebenzisa, izikhala zamabhentshi kufanele zihlanzwe nge-10% sodium hypochlorite (kulandelwe ngamanzi ahlanzekile ukuze kususwe i-bleach esele), i-70% ethanol, noma i-detaminant ebhubhisa i-DNA eqinisekisiwe etholakala emakethe. Okungcono kakhulu, izibani ze-ultra-violet (UV) kufanele zifakwe ukuze kuvunyelwe ukususwa kokungcola ngokukhanya. Kodwa-ke, ukusetshenziswa kwezibani ze-UV kufanele kukhawulelwe ezindaweni zokusebenza ezivaliwe, isibonelo amakhabethe okuphepha, ukuze kuncishiswe ukuvezwa kwe-UV kwabasebenzi belabhoratri. Sicela ulandele imiyalelo yomkhiqizi yokunakekelwa kwezibani ze-UV, umoya kanye nokuhlanza ukuze kuqinisekiswe ukuthi izibani zihlala zisebenza kahle.
Uma usebenzisa i-ethanol engu-70% esikhundleni se-sodium hypochlorite, kuzodingeka imisebe yokukhanya kwe-UV ukuze kuqedwe ukususwa kokungcola.
Ungahlanzi i-vortex kanye ne-centrifuge nge-sodium hypochlorite; esikhundleni salokho, sula nge-ethanol engu-70% bese uyibeka ekukhanyeni kwe-UV, noma usebenzise i-detaminant ebhubhisa i-DNA. Uma kuchithekile, hlola nomkhiqizi ukuze uthole iseluleko esengeziwe sokuhlanza. Uma imiyalelo yomkhiqizi ivuma, ama-pipettes kufanele ahlanzwe njalo nge-autoclave. Uma ama-pipettes engenakuhlanzwa nge-autoclave, kufanele anele ukuwahlanza nge-10% sodium hypochlorite (kulandelwe yi-detaminant ehlanzekile ngamanzi ahlanzekile) noma nge-detaminant ebhubhisa i-DNA ethengiswayo kulandelwe yi-UV.
Ukuhlanza nge-sodium hypochlorite enephesenti eliphezulu kungagcina kulimaze amapulasitiki nezinsimbi ze-pipette uma kwenziwa njalo; hlola izincomo ezivela kumenzi kuqala. Yonke imishini idinga ukulinganiswa njalo ngokwesheduli enconywe ngumkhiqizi. Umuntu oqokiwe kufanele abe ngumphathi wokuqinisekisa ukuthi isheduli yokulinganisa iyalandelwa, izingodo ezinemininingwane ziyagcinwa, futhi amalebula enkonzo aboniswa ngokucacile emishinini.
3. Iseluleko sokusetshenziswa nokuhlanza isikhala sama-molecule esikhethiwe
I-Pre-PCR: Ukulungiswa kwe-Reagent aliquoting / mastermix: Lokhu kufanele kube yindawo ehlanzekile kunazo zonke ezisetshenziselwa ukulungiselela ukuhlolwa kwama-molecule futhi kufanele kube ikhabhinethi yokugeleza ye-laminar ekhethiwe efakwe ukukhanya kwe-UV. Amasampula, i-nucleic acid ekhishwe kanye nemikhiqizo ye-PCR ethuthukisiwe akumele iphathwe kule ndawo. Ama-reagent e-Amplification kufanele agcinwe efrijini (noma esiqandisini, ngokwezincomo zomkhiqizi) endaweni efanayo ekhethiwe, mhlawumbe eduze kwekhabhinethi yokugeleza ye-laminar noma indawo ye-pre-PCR. Amagilavu kufanele ashintshwe isikhathi ngasinye lapho ungena endaweni ye-pre-PCR noma ikhabhinethi yokugeleza ye-laminar.
Indawo yangaphambi kwe-PCR noma ikhabhinethi yokugeleza kwe-laminar kufanele ihlanzwe ngaphambi nangemva kokusetshenziswa kanje: Sula zonke izinto ezikukhabhinethi, isib. ama-pipettes, amabhokisi e-tip, i-vortex, i-centrifuge, ama-tube racks, amapeni, njll. nge-ethanol engu-70% noma i-decontaminant yezentengiselwano ebhubhisa i-DNA, bese uyivumela yome. Endabeni yendawo yokusebenza evaliwe, isib. ikhabhinethi yokugeleza kwe-laminar, beka i-hood ekukhanyeni kwe-UV imizuzu engama-30.
Inothi
Ungazivezi izithasiselo ekukhanyeni kwe-UV; zifake kuphela ekhabetheni uma selihlanzekile. Uma wenza i-PCR yokubhala kabusha, kungase kube usizo ukusula izindawo nemishini ngesisombululo esiphula ama-RNases lapho exhumana. Lokhu kungasiza ekugwemeni imiphumela emibi engamanga evela ekuwohlokeni kwe-enzyme ye-RNA. Ngemva kokuhlanza nangaphambi kokulungisa i-mastermix, amagilavu kufanele ashintshwe futhi, bese ikhabethe selilungele ukusetshenziswa.
I-Pre-PCR: Ukukhishwa kwe-Nucleic acid/ukwengezwa kwethempulethi:
I-Nucleic acid kumele ikhishwe futhi iphathwe endaweni yesibili eqokiwe, kusetshenziswa isethi ehlukile yama-pipettes, amathiphu okuhlunga, ama-tube racks, amagilavu amasha, ama-lab coats kanye neminye imishini. Le ndawo futhi ingeyokwengeza ithempulethi, izilawuli kanye nemigqa yethrendi kuma-tubes noma ama-plate e-mastermix. Ukuze ugweme ukungcoliswa kwamasampula e-nucleic acid akhishwe ahlaziywayo, kunconywa ukushintsha amagilavu ngaphambi kokuphatha izilawuli noma amazinga amahle nokusebenzisa isethi ehlukile yama-pipettes. Ama-reagent e-PCR kanye nemikhiqizo ekhulisiwe akumele ifakwe amapayipi kule ndawo. Amasampula kufanele agcinwe eziqandisini noma eziqandisini eziqokiwe endaweni efanayo. Indawo yokusebenza yesampula kufanele ihlanzwe ngendlela efanayo nendawo ye-mastermix.
I-Post-PCR: Ukukhulisa nokuphatha umkhiqizo okhulisiwe
Lesi sikhala esibekiwe senzelwe izinqubo zangemva kokukhulisa futhi kufanele sihlukaniswe ngokomzimba nezindawo zangaphambi kokukhulisa i-PCR. Ngokuvamile siqukethe ama-thermocycler kanye namapulatifomu esikhathi sangempela, futhi kufanele sibe nekhabhinethi yokugeleza ye-laminar yokwengeza umkhiqizo we-round 1 PCR ku-round 2 reaction, uma kwenziwa i-PCR enezidleke. Ama-reagent e-PCR kanye ne-nucleic acid ekhishwe akumele kuphathwe kule ndawo ngoba ingozi yokungcola iphezulu. Le ndawo kufanele ibe nesethi ehlukile yamagilavu, amajazi elebhu, ama-plate nama-tube racks, ama-pipettes, amathiphu okuhlunga, ama-bin kanye neminye imishini. Ama-tubes kumele afakwe i-centrifuge ngaphambi kokuvula. Indawo yokusebenza yesampula kufanele ihlanzwe ngendlela efanayo nendawo ye-mastermix.
I-Post-PCR: Ukuhlaziywa komkhiqizo
Leli gumbi lenzelwe imishini yokuthola umkhiqizo, isib. amathangi e-gel electrophoresis, amaphakethe kagesi, i-UV transilluminator kanye nohlelo lokubhala imibhalo ye-gel. Le ndawo kufanele ibe namasethi ahlukene amagilavu, amajazi elebhu, ama-plate nama-tube racks, ama-pipettes, amathiphu okuhlunga, ama-bin kanye neminye imishini. Awekho amanye ama-reagent angalethwa kule ndawo, ngaphandle kodayi wokulayisha, i-molecular marker kanye ne-agarose gel, kanye nezingxenye ze-buffer. Indawo yokusebenza yesampula kufanele ihlanzwe ngendlela efanayo nesikhala se-mastermix.
Inothi elibalulekile
Okungcono kakhulu, amakamelo angaphambi kwe-PCR akufanele angene ngosuku olufanayo uma umsebenzi usuvele wenziwe emakamelweni angemva kwe-PCR. Uma lokhu kungenakugwenywa ngokuphelele, qiniseka ukuthi izandla ziqale zigezwe kahle nokuthi amajazi athile elebhu agqokwa emakamelweni. Izincwadi zelebhu namaphepha akufanele kuthathwe kumakamelo angaphambi kwe-PCR uma zisetshenziswe emakamelweni angemva kwe-PCR; uma kudingeka, thatha okuphrintiwe okuphindwe kabili kwezinqubo/ama-ID esampula, njll.
4. Iseluleko esijwayelekile sebhayoloji yama-molecule
Sebenzisa amagilavu angenampuphu ukuze ugweme ukuvimbela ukuhlolwa. Indlela efanele yokufaka amapayipi ibaluleke kakhulu ekunciphiseni ukungcola. Ukufaka amapayipi ngendlela engalungile kungabangela ukuchelela lapho kukhishwa uketshezi kanye nokudalwa kwama-aerosol. Umkhuba omuhle wokufaka amapayipi ngendlela efanele ungatholakala kulezi zixhumanisi ezilandelayo: Umhlahlandlela we-Gilson wokufaka amapayipi, amavidiyo e-Anachem technique pipetting, amashubhu e-Centrifuge ngaphambi kokuvula, bese uwavula ngokucophelela ukuze ugweme ukuchelela. Vala amashubhu ngokushesha ngemva kokusebenzisa ukuze ugweme ukungenisa ukungcola.
Uma wenza ukusabela okuningi, lungisa i-mastermix eyodwa equkethe ama-reagent avamile (isb. amanzi, ama-dNTP, ama-buffer, ama-primer kanye ne-enzyme) ukuze unciphise inani lokudluliselwa kwama-reagent futhi unciphise usongo lokungcola. Kunconywa ukusetha i-mastermix eqhweni noma ebhulokini elibandayo. Ukusetshenziswa kwe-enzyme ye-Hot Start kungasiza ekunciphiseni ukukhiqizwa kwemikhiqizo engaqondile. Vikela ama-reagent aqukethe ama-fluorescent probes ekukhanyeni ukuze ugweme ukuwohloka.
5. Izilawuli zangaphakathi
Faka izilawuli ezinhle nezingalungile ezichazwe kahle, eziqinisekisiwe, kanye nokulawula okungenathempulethi kuzo zonke izimpendulo, kanye nomugqa wethrendi onamaphoyinti amaningi wokusabela okunobuningi. Ukulawula okuhle akufanele kube namandla kangangokuthi kubeka engcupheni yokungcola. Faka izilawuli zokukhipha ezinhle nezingalungile lapho wenza ukukhipha i-nucleic acid.
Kunconywa ukuthi kufakwe imiyalelo ecacile endaweni ngayinye ukuze abasebenzisi bazi imithetho yokuziphatha. Amalabhorethri okuxilonga athola amazinga aphansi kakhulu e-DNA noma i-RNA kumasampula emitholampilo angase afune ukusebenzisa isilinganiso esengeziwe sokuphepha sokuba nezinhlelo zokuphatha umoya ezihlukene ezinengcindezi yomoya ephansi emakamelweni angaphambi kwe-PCR kanye nengcindezi yomoya ephansi emakamelweni angemva kwe-PCR.
Okokugcina, ukuthuthukisa uhlelo lokuqinisekisa ikhwalithi (QA) kuyasiza. Uhlelo olunjalo kufanele lufake uhlu lwamasheya amakhulu e-reagent kanye namasheya asebenzayo, imithetho yokugcina amakhithi kanye nama-reagent, ukubika ngemiphumela yokulawula, izinhlelo zokuqeqesha abasebenzi, ama-algorithms okuxazulula izinkinga, kanye nezinyathelo zokulungisa uma kudingeka.
6. Uhlu lwezincwadi
U-Aslan A, uKinzelman J, uDreelin E, u-Anan'eva T, uLavander J. Isahluko 3: Ukusetha ilabhorethri ye-qPCR. Umbhalo oqondisayo wokuhlola amanzi okuzijabulisa kusetshenziswa indlela ye-USEPA qPCR 1611. ILansing- Michigan State University.
I-Public Health England, i-NHS. Izindinganiso zase-UK zocwaningo lwe-microbiology: Umkhuba Omuhle Welabhorethri lapho kwenziwa izivivinyo zokukhulisa ama-molecule). Isiqondiso Sekhwalithi. 2013;4(4):1–15.
UMifflin T. Ukusetha ilabhorethri ye-PCR. I-Cold Spring Harb Protoc. 2007;7.
Schroeder S 2013. Ukugcinwa njalo kwama-centrifuge: ukuhlanza, ukugcinwa kanye nokubulala amagciwane kwama-centrifuge, ama-rotor kanye nama-adapter (Iphepha elimhlophe No. 14). Hamburg: Eppendorf; 2013.
UViana RV, eWallis CL. Umkhuba Omuhle Welabhorethri Yokwelapha (i-GCLP) wokuhlolwa okusekelwe kuma-molecule okusetshenziswa emalabhorethri okuxilonga, Ku: Akyar I, umhleli. I-spectra ebanzi yokulawula ikhwalithi. I-Rijeka, eCroatia: Intech; 2011: 29–52.
Isikhathi sokuthunyelwe: Julayi-16-2020
