Hanyoyin gano kwayoyin halitta suna da ikon samar da adadi mai yawa na acid nucleic ta hanyar haɓaka adadin da aka samo a cikin samfurori.Duk da yake wannan yana da fa'ida don ba da damar gano mai hankali, yana kuma gabatar da yuwuwar kamuwa da cuta ta hanyar yaduwar iska mai ƙarfi a cikin yanayin dakin gwaje-gwaje.Lokacin gudanar da gwaje-gwaje, za a iya ɗaukar matakan don guje wa gurɓacewar reagents, kayan aikin dakin gwaje-gwaje da sararin benci, saboda irin wannan gurbatar na iya haifar da sakamako mara kyau (ko na ƙarya).

Don taimakawa rage yuwuwar kamuwa da cuta, ya kamata a yi amfani da Kyawawan Ayyukan Laboratory a kowane lokaci.Musamman, ya kamata a yi taka tsantsan game da abubuwa masu zuwa:

1. Magance reagents
2. Ƙungiya na wuraren aiki da kayan aiki
3. Yi amfani da shawarwarin tsaftacewa don wurin da aka keɓe na kwayoyin halitta
4. Gabaɗaya nasihar ilimin halitta
5. Gudanar da ciki
6. Littafi Mai Tsarki

1. Magance reagents

A taƙaice centrifuge reagent bututu kafin buɗewa don guje wa haɓakar iska.Aliquot reagents don guje wa daskare-narkewa da yawa da gurɓatar hannun jari.A sarari yi alama da kwanan wata duk reagents da dauki bututu da kuma kula da rajistan ayyukan reagent yawa da batch lambobi amfani a duk gwaje-gwaje.Pipette duk reagents da samfurori ta amfani da shawarwarin tacewa.Kafin siyan, yana da kyau a tabbatar tare da masana'anta cewa tukwici masu tacewa sun dace da alamar pipette da za a yi amfani da su.

2. Ƙungiya na wuraren aiki da kayan aiki

Ya kamata a tsara wuraren aiki don tabbatar da cewa aikin yana gudana a hanya ɗaya, daga wurare masu tsabta (pre-PCR) zuwa wuraren datti (bayan PCR).Hakkoki na gabaɗaya masu zuwa zasu taimaka don rage yiwuwar kamuwa da cuta.Yi ɗakunan da aka keɓe daban-daban, ko aƙalla wurare daban-daban na zahiri, don: shiri na mastermix, haɓakar acid nucleic da ƙari samfurin DNA, haɓakawa da sarrafa haɓakar samfur, da nazarin samfur, misali gel electrophoresis.

A wasu saitunan, samun dakuna daban 4 yana da wahala.Zaɓuɓɓuka mai yuwuwa amma mafi ƙarancin kyawawa shine yin shiri na mastermix a cikin yanki mai ɗaukar nauyi, misali madaidaicin madaidaicin laminar.A cikin yanayin haɓakawa na PCR na gida, ya kamata a shirya shirye-shiryen mastermix don amsawar zagaye na biyu a cikin yanki mai tsabta don shirye-shiryen mastermix, amma inoculation tare da samfuran PCR na farko yakamata a yi a cikin ɗakin ƙarawa, kuma idan zai yiwu. a cikin keɓantaccen wurin da aka keɓe (misali ma'ajin kwararar laminar).

Kowane ɗaki / yanki yana buƙatar keɓan saiti na bututun mai lakabi a sarari, tukwici masu tacewa, ɗigon bututu, vortexes, centrifuges (idan ya dace), alƙalami, manyan kayan aikin lab, riguna da akwatunan safofin hannu waɗanda za su kasance a wuraren aikinsu.Dole ne a wanke hannaye kuma a canza safar hannu da riguna na lab yayin motsi tsakanin wuraren da aka keɓe.Kada a motsa reagents da kayan aiki daga wuri mai datti zuwa wuri mai tsabta.Idan wani matsanancin hali ya taso inda ake buƙatar mayar da reagent ko yanki na kayan aiki baya, dole ne a fara gurɓata shi da 10% sodium hypochlorite, sannan a shafe shi da ruwa maras kyau.

Lura

Maganin 10% sodium hypochlorite dole ne a yi sabo kowace rana.Lokacin da aka yi amfani da shi don ƙazanta, ya kamata a kiyaye mafi ƙarancin lokacin hulɗa na mintuna 10.
A madadin, ana iya amfani da samfuran kasuwanci waɗanda aka inganta azaman DNA mai lalata saman ƙasa idan shawarwarin aminci na gida ba su ba da izinin amfani da sodium hypochlorite ba ko kuma idan sodium hypochlorite bai dace da lalata sassan ƙarfe na kayan aiki ba.

Da kyau, ya kamata ma'aikata su bi ka'idodin tafiyar da aiki na unidirectional kuma kada su fita daga wuraren datti (bayan PCR) zuwa wuraren tsabta (pre-PCR) a rana guda.Duk da haka, ana iya samun lokatai da wannan ba zai yuwu ba.Lokacin da irin wannan taron ya taso, dole ne ma'aikata su kula da wanke hannu sosai, canza safar hannu, yin amfani da rigar da aka keɓance kuma kada su sake gabatar da wani kayan aiki da za su sake fitar da su daga ɗakin, kamar littattafan lab.Ya kamata a jaddada irin waɗannan matakan kulawa a cikin horar da ma'aikata game da hanyoyin kwayoyin.

Bayan amfani, ya kamata a tsaftace wuraren benci tare da 10% sodium hypochlorite (bayan ruwa mai tsabta don cire ragowar bleach), 70% ethanol, ko ingantacciyar hanyar sayar da DNA mai lalata lalata.Da kyau, ya kamata a sanya fitilun ultraviolet (UV) don ba da damar lalata ta hanyar sakawa.Koyaya, yakamata a iyakance amfani da fitilun UV zuwa wuraren aiki da aka rufe, misali kabad ɗin tsaro, don iyakance tasirin UV na ma'aikatan dakin gwaje-gwaje.Da fatan za a bi umarnin masana'anta don kula da fitilar UV, samun iska da tsaftacewa don tabbatar da cewa fitulun sun kasance masu tasiri.

Idan amfani da 70% ethanol maimakon sodium hypochlorite, za a buƙaci sakawa tare da hasken UV don kammala lalata.
Kada a tsaftace vortex da centrifuge tare da sodium hypochlorite;a maimakon haka, shafa ƙasa da 70% ethanol da fallasa zuwa hasken UV, ko amfani da lalatawar DNA na kasuwanci.Don zubewa, duba tare da masana'anta don ƙarin shawarwarin tsaftacewa.Idan umarnin masana'anta ya ba da izini, ya kamata a ba wa pipettes haifuwa akai-akai ta autoclave.Idan pipettes ba za a iya sarrafa su ba, ya isa ya tsaftace su da 10% sodium hypochlorite (biye da cikakken gogewa tare da ruwa mara kyau) ko tare da lalata DNA mai lalata DNA wanda ke biye da bayyanar UV.

Tsaftacewa tare da babban adadin sodium hypochlorite na iya lalata robobin pipette da karafa idan an yi akai-akai;duba shawarwari daga masana'anta da farko.Duk kayan aikin suna buƙatar a daidaita su akai-akai bisa ga tsarin da masana'anta suka ba da shawarar.Mutumin da aka zaɓa ya kamata ya kasance mai kula da tabbatar da cewa an kiyaye jadawalin daidaitawa, ana kiyaye cikakkun bayanai, kuma ana nuna alamun sabis akan kayan aiki.

3. Yi amfani da shawarwarin tsaftacewa don wurin da aka keɓe na kwayoyin halitta

Pre-PCR: Reagent aliquoting / mastermix shiri: Wannan yakamata ya zama mafi tsaftar duk wuraren da aka yi amfani da shi don shirye-shiryen gwaje-gwajen ƙwayoyin cuta kuma yakamata ya zama ƙayyadaddun majalisar da aka keɓe na kwararar laminar sanye take da hasken UV.Samfurori, fitar da acid nucleic da ingantattun samfuran PCR ba dole ba ne a sarrafa su a wannan yanki.Ya kamata a adana reagents na haɓakawa a cikin injin daskarewa (ko firiji, kamar yadda shawarwarin masana'anta) a cikin sararin da aka keɓance, da kyau kusa da ma'aunin wutar lantarki ko yankin pre-PCR.Ya kamata a canza safar hannu kowane lokaci yayin shigar da yankin pre-PCR ko majalisar kula da kwararar laminar.

Ya kamata a tsaftace yankin pre-PCR ko ma'ajin ruwan laminar kafin da kuma bayan amfani kamar haka: Goge duk abubuwan da ke cikin majalisar, misali pipettes, akwatunan tip, vortex, centrifuge, racks tube, alkaluma, da sauransu tare da 70% ethanol ko DNA na kasuwanci mai lalata ƙazanta, da ba da damar bushewa.A cikin yanayin rufaffiyar wurin aiki, misali madaidaicin madaidaicin laminar, fallasa murfin ga hasken UV na mintuna 30.

Lura

Kada a bijirar da reagents zuwa hasken UV;kawai matsar da su a cikin majalisar da zarar ya tsarkaka.Idan yin juzu'i na PCR, yana iya zama taimako don goge saman da kayan aiki tare da maganin da ke rushe RNases akan lamba.Wannan na iya taimakawa don guje wa sakamakon karya-mara kyau daga lalatawar enzyme na RNA.Bayan lalatawa da kuma kafin shirya mastermix, safofin hannu ya kamata a sake canza safofin hannu, sannan majalisar ta shirya don amfani.

Pre-PCR: Nucleic acid hakar/haɗin samfur:

Dole ne a fitar da acid nucleic kuma a sarrafa shi a cikin wani yanki na biyu da aka keɓance, ta yin amfani da saitin pipettes daban-daban, tukwici masu tacewa, raƙuman bututu, sabbin safofin hannu, riguna na lab da sauran kayan aiki.Wannan yanki kuma don ƙari ne na samfuri, sarrafawa da yanayin yanayin zuwa ga mastermix tubes ko faranti.Don guje wa gurɓata samfuran nucleic acid da aka fitar waɗanda ake nazari, ana ba da shawarar a canza safar hannu kafin sarrafa ingantattun sarrafawa ko ƙa'idodi da amfani da wani sashe na pipettes.Dole ne ba za a yi amfani da reagents na PCR da haɓakar samfuran a wannan yanki ba.Ya kamata a adana samfurori a cikin firji da aka keɓe ko daskarewa a wuri ɗaya.Ya kamata a tsaftace wurin aikin samfurin kamar yadda sararin mastermix yake.

Post-PCR: Ƙarawa da sarrafa kayan haɓakawa

Wannan sarari da aka keɓance don matakan haɓakawa ne kuma yakamata ya zama keɓance a zahiri daga wuraren pre-PCR.Yawanci yana ƙunshi masu amfani da injin motsa jiki da dandamali na lokaci-lokaci, kuma yakamata ya kasance yana da madaidaicin ma'auni don ƙara samfurin PCR na zagaye 1 zuwa matakin zagaye na 2, idan ana yin PCR na gida.Dole ne a kula da reagents na PCR da acid nucleic acid a wannan yanki tunda haɗarin kamuwa da cuta yana da yawa.Wannan yanki ya kamata ya kasance yana da safofin hannu daban-daban, riguna na lab, faranti da rakuman bututu, pipettes, tukwici masu tacewa, kwanduna da sauran kayan aiki.Tubes dole ne a sanya su a tsakiya kafin buɗewa.Ya kamata a tsaftace wurin aikin samfurin kamar yadda sararin mastermix yake.

Post-PCR: Binciken samfur

Wannan ɗakin don kayan aikin gano samfur ne, misali tankunan lantarki na gel electrophoresis, fakitin wutar lantarki, transilluminator UV da tsarin takaddun gel.Wannan yanki yakamata ya kasance yana da safofin hannu daban-daban, riguna na lab, faranti da tagulla, pipettes, tukwici na tacewa, kwanduna da sauran kayan aiki.Ba za a iya shigar da wasu reagents cikin wannan yanki ba, ban da rini mai ɗaukar nauyi, alamar kwayoyin halitta da gel agarose, da abubuwan buffer.Ya kamata a tsaftace wurin aikin samfurin kamar yadda sararin mastermix yake.

Bayani mai mahimmanci

Da kyau, kada a shigar da ɗakunan pre-PCR a rana ɗaya idan an riga an yi aiki a ɗakunan bayan PCR.Idan wannan ya kasance ba makawa gaba ɗaya, tabbatar da cewa an fara wanke hannaye sosai kuma an sanya takamaiman riguna na lab a cikin ɗakuna.Ba dole ba ne a shigar da littattafan Lab da takarda a cikin ɗakunan pre-PCR idan an yi amfani da su a ɗakunan bayan PCR;idan ya cancanta, ɗauki kwafin-fitin ladabi/samfuran ID, da sauransu.

4. Gabaɗaya nasihar ilimin halitta

Yi amfani da safar hannu marasa foda don guje wa hanawa tantancewa.Ingantacciyar dabarar bututu ita ce mafi mahimmanci don rage gurɓatawa.Bututun da ba daidai ba zai iya haifar da fantsama yayin rarraba ruwa da ƙirƙirar iska.Ana iya samun kyakkyawan aiki don daidaitaccen bututu a hanyoyin haɗin yanar gizo masu zuwa: Jagorar Gilson zuwa bututu, Bidiyon fasaha na fasaha na Anachem, Bututun Centrifuge kafin buɗewa, kuma buɗe su a hankali don guje wa fantsama.Rufe bututu nan da nan bayan amfani don guje wa gabatarwar gurɓataccen abu.

Lokacin aiwatar da halayen da yawa, shirya mastermix ɗaya wanda ke ɗauke da reagents gama gari (misali ruwa, dNTPs, buffer, primers da enzyme) don rage yawan canja wurin reagent da rage barazanar gurɓatawa.Ana bada shawara don saita mastermix akan kankara ko shinge mai sanyi.Yin amfani da enzyme mai zafi mai zafi na iya taimakawa wajen rage samar da samfuran da ba na musamman ba.Kare reagents masu ƙunshe da na'urorin kyalli daga haske don gujewa lalacewa.

5. Gudanar da ciki

Haɗa da siffa mai kyau, tabbataccen sarrafawa masu inganci da mara kyau, tare da mara ƙima a cikin duk halayen, da madaidaitan ma'auni mai yawa don halayen ƙididdiga.Kyakkyawan iko bai kamata ya kasance mai ƙarfi sosai har ya haifar da haɗarin kamuwa da cuta ba.Haɗa ingantattun sarrafawar cirewa mara kyau yayin aiwatar da hakar acid nucleic.

Ana ba da shawarar cewa a buga takamaiman umarni a kowane yanki don masu amfani su san ƙa'idodin ɗabi'a.Dakunan gwaje-gwaje masu gano ƙananan matakan DNA ko RNA a cikin samfuran asibiti na iya son ɗaukar ƙarin ma'aunin tsaro na samun tsarin sarrafa iska daban tare da ɗan ƙaramin iska mai inganci a cikin ɗakunan pre-PCR da ƙarancin iska mara kyau a cikin ɗakunan bayan PCR.

A ƙarshe, haɓaka shirin tabbatar da inganci (QA) yana da taimako.Irin wannan shirin ya kamata ya haɗa da jerin sunayen manyan hannun jari na reagent da hannun jari masu aiki, dokoki don adana kayan aiki da reagents, rahoton sakamakon sarrafawa, shirye-shiryen horar da ma'aikata, algorithms warware matsala, da ayyukan gyara lokacin da ake buƙata.

6. Littafi Mai Tsarki

Aslan A, Kinzelman J, Dreelin E, Anan'eva T, Lavander J. Babi na 3: Kafa dakin gwaje-gwaje na qPCR.Takardun jagora don gwada ruwan nishaɗi ta amfani da USEPA qPCR hanyar 1611. Lansing-Jami'ar Jihar Michigan.

Kiwon Lafiyar Jama'a Ingila, NHS.Matsayin Burtaniya don binciken ƙananan ƙwayoyin cuta: Kyawun Ƙwararrun Ƙwararrun Ƙwararrun Ƙwararrun Ƙwararrun Ƙwararrun Ƙwararrun Ƙwararrun Ƙwararrun Ƙwararrun Ƙwararru).Kyakkyawan Jagora.2013; 4 (4): 1–15.

Miffin T. Kafa dakin gwaje-gwaje na PCR.Cold Spring Harb Protoc.2007; 7.

Schroeder S 2013. Kulawa na yau da kullun na centrifuges: tsaftacewa, kulawa da disinfection na centrifuges, rotors da adaftan (Fara takarda No. 14).Hamburg: Eppendorf;2013.

Viana RV, Wallis CL.Kyawawan Ayyukan Laboratory Clinical (GCLP) don gwajin tushen kwayoyin halitta da aka yi amfani da su a dakunan gwaje-gwaje, A: Akyar I, edita.Faɗin bakan na kula da inganci.Rijeka, Croatia: Fasaha;2011: 29-52.