Iindlela zokufumanisa iimolekyuli zinamandla okuvelisa umthamo omkhulu we-nucleic acid ngokwandisa ubungakanani be-trace efunyenwe kwiisampuli. Nangona oku kuluncedo ekuvumeleni ukufunyanwa okunobuthathaka, kukwazisa nokuba kungenzeka ukungcola ngokusasazwa kwee-aerosols zokwandisa kwindawo yelabhoratri. Xa kusenziwa uvavanyo, amanyathelo anokuthathwa ukuze kuthintelwe ukungcola kwee-reagents, izixhobo zelabhoratri kunye nendawo yebhentshi, njengoko ukungcola okunjalo kunokuvelisa iziphumo ezingezizo (okanye ezingezizo).
Ukunceda ekunciphiseni amathuba ongcoliseko, kufuneka kusetyenziswe iMisebenzi yeLabhoratri elungileyo ngamaxesha onke. Ngokukodwa, kufuneka kuthathwe amanyathelo okhuseleko malunga nala manqaku alandelayo:
1. Ukuphatha ii-reagents
2. Ulungelelwaniso lwendawo yokusebenza kunye nezixhobo
3. Ingcebiso yokusebenzisa nokucoca indawo ekhethiweyo yeemolekyuli
4. Ingcebiso ngokubanzi yebhayoloji yemolekyuli
5. Ulawulo lwangaphakathi
6. Uluhlu lweencwadi
1. Ukuphatha ii-reagents
Okwethutyana faka iityhubhu ze-reagent ze-centrifuge ngaphambi kokuba zivulwe ukuze kuthintelwe ukuveliswa kwee-aerosols. Beka ii-reagents ukuze kuthintelwe ukunyibilika okuninzi kunye nongcoliseko lwesitokhwe esiphambili. Bhala ilebheli kwaye ubeke umhla kuzo zonke iityhubhu ze-reagent kunye ne-reaction kwaye ugcine iilog zenani le-reagent kunye neenombolo zebhetshi ezisetyenzisiweyo kuzo zonke iimvavanyo. Faka ii-reagents kunye neesampuli usebenzisa iingcebiso zesihluzi. Ngaphambi kokuthenga, kuyacetyiswa ukuba uqinisekise nomenzi ukuba iingcebiso zesihluzi zihambelana nophawu lwepipette eza kusetyenziswa.
2. Ulungelelwaniso lwendawo yokusebenza kunye nezixhobo
Indawo yokusebenza mayicwangciswe ukuqinisekisa ukuba ukuhamba komsebenzi kwenzeka kwicala elinye, ukusuka kwiindawo ezicocekileyo (pre-PCR) ukuya kwiindawo ezingcolileyo (post-PCR). La manyathelo alandelayo aqhelekileyo aya kunceda ukunciphisa amathuba okungcola. Yiba namagumbi ahlukeneyo akhethiweyo, okanye ubuncinane kwiindawo ezahlukeneyo ngokwasemzimbeni, ukulungiselela: i-mastermix, ukukhupha i-nucleic acid kunye nokongezwa kwethempleyithi ye-DNA, ukukhulisa nokuphatha imveliso eyongeziweyo, kunye nohlalutyo lwemveliso, umz. i-gel electrophoresis.
Kwezinye iimeko, ukuba namagumbi amane ahlukeneyo kunzima. Ukhetho olunokwenzeka kodwa olungathandekiyo kukwenza amalungiselelo e-mastermix kwindawo yokuthintela, umz. ikhabhinethi yokuhamba kwe-laminar. Kwimeko yokukhulisa i-PCR eneentaka, ukulungiswa kwe-mastermix yokusabela kwesibini kufuneka kulungiselelwe kwindawo 'ecocekileyo' yokulungiselela i-mastermix, kodwa ukugonywa ngemveliso ye-PCR ephambili kufuneka kwenziwe kwigumbi lokukhulisa, kwaye ukuba kunokwenzeka kwindawo yokuthintela ekhethekileyo (umz. ikhabhinethi yokuhamba kwe-laminar).
Igumbi/indawo nganye ifuna iseti eyahlukileyo yeepipettes ezibhalwe ngokucacileyo, iincam zesihluzi, ii-tube racks, ii-vortexes, ii-centrifuges (ukuba zifanelekile), iipeni, ii-generic lab reagents, ii-lab coats kunye neebhokisi zeeglavu eziza kuhlala kwiindawo zazo zokusebenza. Izandla mazihlanjwe kwaye iiglavu kunye nee-lab coats zitshintshwe xa kushukunyiswa phakathi kweendawo ezimiselweyo. Ii-reagents kunye nezixhobo akufuneki zisuswe kwindawo engcolileyo ziye kwindawo ecocekileyo. Ukuba kuvela imeko egqithisileyo apho i-reagent okanye isixhobo kufuneka sibuyiselwe ngasemva, kufuneka kuqala sisuswe kwi-10% ye-sodium hypochlorite, kulandele ukusula ngamanzi acocekileyo.
Phawula
Isisombululo se-10% sodium hypochlorite kufuneka senziwe sitsha yonke imihla. Xa sisetyenziselwa ukucoca ukungcola, kufuneka kulandelwe ubuncinane ixesha lokudibana ne-10 imizuzu.
Kungenjalo, iimveliso ezithengiswayo eziqinisekisiweyo njengezinto zokucoca umphezulu ezitshabalalisa i-DNA zingasetyenziswa ukuba iingcebiso zokhuseleko lwendawo azivumeli ukusetyenziswa kwe-sodium hypochlorite okanye ukuba i-sodium hypochlorite ayifanelekanga ukucoca iindawo zesinyithi zezixhobo.
Okona kulungileyo kukuba, abasebenzi bafanele balandele indlela yokusebenza ehambelanayo kwaye bangasuki kwiindawo ezingcolileyo (emva kwe-PCR) babuyele kwiindawo ezicocekileyo (emva kwe-PCR) ngaloo mini inye. Nangona kunjalo, kunokubakho amaxesha apho oku kungenakuphepheka. Xa kuvela imeko enjalo, abasebenzi kufuneka baqaphele ukuhlamba izandla ngokupheleleyo, batshintshe iiglavu, basebenzise ijazi laboratory elimiselweyo kwaye bangangenisi naziphi na izixhobo abaza kufuna ukuzikhupha kwigumbi kwakhona, njengeencwadi zelebhu. Amanyathelo olawulo anjalo kufuneka agxininiswe kuqeqesho lwabasebenzi ngeendlela zemolekyuli.
Emva kokusetyenziswa, izithuba zeebhentshi mazicocwe nge-10% ye-sodium hypochlorite (kulandele amanzi angenazintsholongwane ukususa i-bleach eseleyo), i-70% ye-ethanol, okanye i-DNA-destroying detaminant eqinisekisiweyo ethengiswayo. Okona kulungileyo, kufuneka kufakelwe izibane ze-ultra-violet (UV) ukuze kukwazi ukususwa kongcoliseko ngokukhanya. Nangona kunjalo, ukusetyenziswa kwezibane ze-UV kufuneka kuthintelwe kwiindawo zokusebenza ezivaliweyo, umz. iikhabhathi zokhuseleko, ukuze kuthintelwe ukuvezwa kwe-UV kwabasebenzi belebhu. Nceda ulandele imiyalelo yomenzi yokhathalelo lwezibane ze-UV, umoya kunye nokucoca ukuqinisekisa ukuba izibane zihlala zisebenza.
Ukuba usebenzisa i-70% ye-ethanol endaweni ye-sodium hypochlorite, kuya kufuneka ukukhanyiswa nge-UV light ukuze kugqitywe ukususwa kongcoliseko.
Musa ukucoca i-vortex kunye ne-centrifuge nge-sodium hypochlorite; endaweni yoko, sula nge-70% ye-ethanol uze uyibeke ekukhanyeni kwe-UV, okanye usebenzise i-detergent etshabalalisa i-DNA. Ukuba kukho ukuchitheka, jonga kumenzi ukuze ufumane iingcebiso ezithe vetshe zokucoca. Ukuba imiyalelo yomenzi iyavuma, ii-pipettes kufuneka zihlanjululwe rhoqo nge-autoclave. Ukuba ii-pipettes azinakuhlanjululwa nge-autoclave, kufanele ukuba zanele ukuzicoca nge-10% ye-sodium hypochlorite (kulandelwe yi-detergent ecocekileyo ngamanzi acocekileyo) okanye nge-detergent etshabalalisa i-DNA ethengiswayo kulandelwe yi-UV exposure.
Ukucoca nge-sodium hypochlorite ephezulu kunokonakalisa iiplastiki kunye neentsimbi zepipette ukuba kwenziwa rhoqo; jonga iingcebiso ezivela kumenzi kuqala. Zonke izixhobo kufuneka zilinganiswe rhoqo ngokweshedyuli ecetyiswa ngumenzi. Umntu otyunjiweyo kufuneka abe noxanduva lokuqinisekisa ukuba ishedyuli yokulinganisa iyalandelwa, iirekhodi ezineenkcukacha ziyagcinwa, kwaye iileyibhile zenkonzo ziboniswe ngokucacileyo kwizixhobo.
3. Ingcebiso yokusebenzisa nokucoca indawo ekhethiweyo yeemolekyuli
I-Pre-PCR: Ukulungiswa kwe-Reagent aliquoting / mastermix: Le kufuneka ibe yeyona ndawo icocekileyo kuzo zonke iindawo ezisetyenziselwa ukulungiselela uvavanyo lweemolekyuli kwaye kufanele ukuba ibe yikhabhinethi yokuhamba kwe-laminar ekhethiweyo exhotyiswe ngokukhanya kwe-UV. Iisampuli, i-nucleic acid ekhutshiweyo kunye neemveliso ze-PCR ezikhutshiweyo akufuneki ziphathwe kule ndawo. Ii-reagents zokwandisa kufuneka zigcinwe kwifriji (okanye efrijini, ngokweengcebiso zomenzi) kwindawo enye ekhethiweyo, kungcono ukuba kufutshane nekhabhinethi yokuhamba kwe-laminar okanye indawo ye-pre-PCR. Iiglavu kufuneka zitshintshwe ngalo lonke ixesha xa ungena kwindawo ye-pre-PCR okanye ikhabhinethi yokuhamba kwe-laminar.
Indawo ye-pre-PCR okanye ikhabhinethi yokuhamba kwe-laminar kufuneka icocwe ngaphambi nasemva kokusetyenziswa ngolu hlobo lulandelayo: Sula zonke izinto ezikwikhabhinethi, umz. ii-pipettes, ii-tip box, i-vortex, i-centrifuge, ii-tube racks, ii-pen, njl.njl. nge-70% ethanol okanye i-destroying DNA detaminant yorhwebo, uze uyivumele yome. Kwimeko yendawo yokusebenza evaliweyo, umz. ikhabhinethi yokuhamba kwe-laminar, beka i-hood ekukhanyeni kwe-UV imizuzu engama-30.
Phawula
Musa ukubeka ii-reagents ekukhanyeni kwe-UV; zifake kuphela kwikhabhinethi xa sele icocekile. Ukuba wenza i-PCR yokuguqula i-reverse transcription, kunokuba luncedo ukusula imiphezulu nezixhobo ngesisombululo esiphula ii-RNases xa zidibene. Oku kunokunceda ukuthintela iziphumo ezingezizo ezilungileyo ezivela ekuwohlokeni kwe-enzyme ye-RNA. Emva kokucoca nangaphambi kokulungisa i-mastermix, iiglavu kufuneka zitshintshwe kwakhona, kwaye ikhabhinethi ilungele ukusetyenziswa.
I-Pre-PCR: Ukukhupha i-Nucleic acid/ukongeza itemplate:
I-Nucleic acid kufuneka ikhutshwe kwaye iphathwe kwindawo yesibini ekhethiweyo, kusetyenziswa iseti eyahlukileyo yeepipettes, iincam zesihluzi, iirakhi zeetyhubhu, iiglavu ezintsha, iilabhoratri kunye nezinye izixhobo. Le ndawo ikwayongezwa itemplate, ulawulo kunye neendlela ezihamba ngazo kwiityhubhu okanye iiplate ze-mastermix. Ukuze kuthintelwe ungcoliseko lweesampuli ze-nucleic acid ezikhutshiweyo ezihlalutywayo, kucetyiswa ukuba utshintshe iiglavu ngaphambi kokuphatha ulawulo okanye imigangatho elungileyo kwaye usebenzise iseti eyahlukileyo yeepipettes. Ii-reagents ze-PCR kunye neemveliso ezikhutshiweyo akufuneki zifakwe kwiipayipi kule ndawo. Iisampuli kufuneka zigcinwe kwiifriji okanye kwiifriji ezikhethiweyo kwindawo enye. Indawo yokusebenza yesampuli kufuneka icocwe ngendlela efanayo nendawo ye-mastermix.
I-Post-PCR: Ukwandiswa kunye nokuphathwa kwemveliso ephuculweyo
Le ndawo ibekelwe yona yenzelwe iinkqubo zokwandisa amandla emva kokukhulisa kwaye kufuneka yahlulwe ngokwasemzimbeni kwiindawo zangaphambi kokusebenzisa i-PCR. Ihlala iqulethe ii-thermocyclers kunye namaqonga exesha langempela, kwaye kufanelekile ukuba ibe nekhabhathi yokuhamba kwe-laminar yokongeza imveliso ye-PCR ejikelezayo kwi-reaction ye-round 2, ukuba i-PCR ene-nest iyenziwa. Ii-reagents ze-PCR kunye ne-nucleic acid ekhutshiweyo akufuneki ziphathwe kule ndawo kuba umngcipheko wongcoliseko uphezulu. Le ndawo kufuneka ibe neseti eyahlukileyo yeeglavu, ii-lab coats, ii-plate kunye nee-tube racks, ii-pipettes, ii-filter tips, ii-bins kunye nezinye izixhobo. Ii-tubes kufuneka zifakwe i-centrifuge ngaphambi kokuba zivulwe. Indawo yokusebenza yesampulu kufuneka icocwe ngendlela efanayo nendawo ye-mastermix.
Emva kwePCR: Uhlalutyo lwemveliso
Eli gumbi lenzelwe izixhobo zokuchonga imveliso, umz. iitanki ze-gel electrophoresis, iipakethi zamandla, i-UV transilluminator kunye nenkqubo yoxwebhu lwe-gel. Le ndawo kufuneka ibe neeseti ezahlukeneyo zeeglavu, ii-lab coats, ii-plate kunye nee-tube racks, ii-pipettes, ii-filter tips, ii-bins kunye nezinye izixhobo. Akukho ezinye ii-reagents ezinokuziswa kule ndawo, ngaphandle kwedayi yokulayisha, i-molecular marker kunye ne-agarose gel, kunye nee-buffer components. Indawo yokusebenza yesampulu kufuneka icocwe ngendlela efanayo nendawo ye-mastermix.
Inqaku elibalulekileyo
Eyona nto ingcono kukuba amagumbi angaphambi kwe-PCR angangeniswa ngaloo mini inye ukuba umsebenzi sele wenziwe kumagumbi asemva kwe-PCR. Ukuba oku akunakwenzeka ngokupheleleyo, qiniseka ukuba izandla zihlanjwa kakuhle kuqala kwaye kunxitywe iijazi zelebhu ezithile kumagumbi. Iincwadi kunye namaphepha elabhoratri akufuneki athathwe kumagumbi angaphambi kwe-PCR ukuba asetyenziswe kumagumbi asemva kwe-PCR; ukuba kuyimfuneko, thatha iikopi eziphindwe kabini zeenkqubo/ii-ID zesampulu, njl.
4. Ingcebiso ngokubanzi yebhayoloji yemolekyuli
Sebenzisa iiglavu ezingenamgubo ukuze uphephe ukuthintela uvavanyo. Indlela efanelekileyo yokufaka imibhobho ibaluleke kakhulu ekunciphiseni ungcoliseko. Ukufaka imibhobho ngendlela engalunganga kunokubangela ukutshiza xa kukhutshwa ulwelo kunye nokudalwa kwee-aerosols. Indlela elungileyo yokufaka imibhobho ngendlela echanekileyo inokufumaneka kwezi khonkco zilandelayo: Isikhokelo sikaGilson sokufaka imibhobho, iividiyo zendlela yokufaka imibhobho ye-Anachem, iityhubhu ze-Centrifuge ngaphambi kokuba zivulwe, kwaye uzivule ngononophelo ukuze uphephe ukutshiza. Vala iityhubhu ngokukhawuleza emva kokusetyenziswa ukuze uphephe ukungeniswa kwezinto ezingcolisayo.
Xa usenza ii-reaction ezininzi, lungisa i-mastermix enye equlethe ii-reagents eziqhelekileyo (umz. amanzi, ii-dNTPs, ii-buffer, ii-primers kunye nee-enzyme) ukunciphisa inani lee-reagent transfers kunye nokunciphisa umngcipheko wokungcola. Kucetyiswa ukuba umise i-mastermix kumkhenkce okanye kwi-cold block. Ukusetyenziswa kwe-Hot Start enzyme kunokunceda ukunciphisa imveliso yeemveliso ezingezizo ezithile. Khusela ii-reagents ezinee-fluorescent probes ekukhanyeni ukuze kuthintelwe ukubola.
5. Ulawulo lwangaphakathi
Bandakanya ulawulo olucacileyo noluqinisekisiweyo, kunye nolawulo olungena template kuzo zonke ii-reaction, kunye nomgca we-trend-point titrated wee-reactions zobuninzi. Ulawulo oluhle akufuneki lube namandla kangangokuba lube nomngcipheko wongcoliseko. Bandakanya ulawulo lokukhupha oluhle nolungalunganga xa usenza i-nucleic acid extraction.
Kucetyiswa ukuba kuthunyelwe imiyalelo ecacileyo kwindawo nganye ukuze abasebenzisi bazi ngemithetho yokuziphatha. Iilabhoratri zokuxilonga ezifumanisa amanqanaba aphantsi kakhulu e-DNA okanye i-RNA kwiisampuli zeklinikhi zinokufuna ukwamkela umlinganiselo wokhuseleko owongezelelweyo wokuba neenkqubo zokuphatha umoya ezahlukeneyo ezinoxinzelelo lomoya oluncinci oluhle kumagumbi angaphambi kwe-PCR kunye noxinzelelo lomoya oluncinci olubi kumagumbi asemva kwe-PCR.
Okokugqibela, ukuphuhlisa isicwangciso sokuqinisekisa umgangatho (QA) kuyanceda. Isicwangciso esinjalo kufuneka siquke uluhlu lwesitokhwe esikhulu se-reagent kunye nezitokhwe ezisebenzayo, imithetho yokugcina izixhobo kunye nee-reagents, ingxelo yeziphumo zolawulo, iinkqubo zoqeqesho lwabasebenzi, ii-algorithms zokusombulula iingxaki, kunye namanyathelo okulungisa xa kufuneka.
6. Uluhlu lweencwadi
UAslan A, uKinzelman J, uDreelin E, uAnan'eva T, uLavander J. Isahluko 3: Ukusekwa kwelebhu ye-qPCR. Uxwebhu lwesikhokelo sokuvavanya amanzi okuzonwabisa kusetyenziswa indlela ye-USEPA qPCR 1611. IYunivesithi yaseLansing- Michigan State.
Impilo yoLuntu eNgilane, i-NHS. Imigangatho yase-UK yophando lwe-microbiology: Indlela elungileyo yokusebenza kwelebhu xa kusenziwa uvavanyo lokukhulisa iimolekyuli). Isikhokelo soMgangatho. 2013;4(4):1–15.
UMifflin T. Ukuseta ilebhu ye-PCR. I-Cold Spring Harb Protoc. 2007;7.
USchroeder S 2013. Ukugcinwa rhoqo kwee-centrifuge: ukucoca, ukugcinwa kunye nokubulala iintsholongwane kwii-centrifuge, ii-rotors kunye nee-adapters (Iphepha elimhlophe No. 14). IHamburg: Eppendorf; 2013.
UViana RV, eWallis CL. I-Good Clinical Laboratory Practice (GCLP) yovavanyo olusekelwe kwimolekyuli olusetyenziswa kwiilabhoratri zokuxilonga, Ku: Akyar I, umhleli. Iispectra ezibanzi zolawulo lomgangatho. Rijeka, eCroatia: Intech; 2011: 29–52.
Ixesha lokuposa: Julayi-16-2020
