Ka taea e ngā tikanga kimi ngota te whakaputa i te nui o te waikawa karihi mā te whakanui ake i ngā rahinga iti i kitea i roto i ngā tauira. Ahakoa he pai tēnei mō te āhei i te kimi tairongo, ka whakaurua mai hoki te tūponotanga o te poke mā te horapa o ngā haukoti whakanui i roto i te taiao taiwhanga. I te wā e whakahaere ana i ngā whakamātautau, ka taea te mahi i ngā mahi hei karo i te poke o ngā matū, ngā taputapu taiwhanga me te wāhi tūru, nā te mea ka puta pea he hua teka-pai (he hua teka-kino rānei) mai i taua poke.
Hei āwhina i te whakaiti i te tūponotanga o te poke, me whakamahi i te Tikanga Taiwhanga Pai i ngā wā katoa. Me āta tupato ki ēnei āhuatanga e whai ake nei:
1. Te whakahaere i ngā matū
2. Te whakaritenga o te wāhi mahi me ngā taputapu
3. Ngā tohutohu mō te whakamahinga me te horoi mō te wāhi ngota ngota kua tohua
4. Ngā tohutohu whānui mō te koiora ngota
5. Ngā mana whakahaere ā-roto
6. Rārangi Pukapuka
1. Te whakahaere i ngā matū
Whakapūmautia ngā ngongo matū i mua i te whakatuwheratanga kia kore ai e puta he hau. Whakawhāitihia ngā matū hei karo i te rewa maha me te poke o ngā taonga matua. Tāutuhia mārama ngā tapanga me te rā mō ngā ngongo matū me ngā ngongo tauhohenga katoa, ā, puritia ngā rārangi o ngā tau o te puranga matū me ngā tau puranga i whakamahia i roto i ngā whakamātautau katoa. Whakapūmautia ngā matū me ngā tauira katoa mā te whakamahi i ngā pito tātari. I mua i te hoko, he mea tika kia whakaū ki te kaihanga kei te ōrite ngā pito tātari ki te waitohu o te pipette e whakamahia ana.
2. Te whakaritenga o te wāhi mahi me ngā taputapu
Me whakarite te wāhi mahi kia kotahi te huarahi e rere ai te mahi, mai i ngā wāhi ma (pre-PCR) ki ngā wāhi paru (post-PCR). Mā ēnei mahi ārai whānui e whai ake nei ka āwhina i te whakaiti i te tūponotanga o te poke. Me whakarite he rūma motuhake, he wāhi motuhake rānei, mō: te whakarite i te mastermix, te tango waikawa karihi me te tāpiri tauira DNA, te whakanui me te whakahaere i te hua whakanui, me te tātari hua, hei tauira, te electrophoresis gel.
I ētahi wāhi, he uaua te whai i ngā rūma motuhake e whā. Ko tētahi kōwhiringa pea engari kāore i te tino hiahiatia ko te mahi i te whakarite i te mastermix i roto i tētahi wāhi pupuri, hei tauira, he kāpata rere laminar. Mēnā he whakakaha PCR kōpiko, me whakarite te whakarite o te mastermix mō te tauhohenga tuarua i roto i te wāhi 'ma' mō te whakarite mastermix, engari ko te werohanga ki te hua PCR tuatahi me mahi i roto i te rūma whakakaha, ā, ki te taea i roto i tētahi wāhi pupuri motuhake (hei tauira, he kāpata rere laminar).
Me whai huinga motuhake o ngā pipette, ngā pito tātari, ngā whata ngongo, ngā vortex, ngā centrifuge (mēnā e tika ana), ngā pene, ngā reagents taiwhanga whānui, ngā koti taiwhanga me ngā pouaka karapu ka waiho ki ō rātou teihana mahi. Me horoi ngā ringaringa, ā, me whakakapi ngā karapu me ngā koti taiwhanga ina nekehia ana i waenga i ngā wāhi kua tohua. Kaua ngā reagents me ngā taputapu e nekehia mai i te wāhi paru ki te wāhi ma. Mēnā ka puta he take nui e hiahia ana kia nekehia whakamuri te reagents, te taputapu rānei, me horoi tuatahi ki te 10% konutai hypochlorite, ā, muri iho ka murua ki te wai parakore.
Tuhipoka
Me hanga hou te otinga 10% konutai haikororāiti i ia rā. Mēnā ka whakamahia hei horoi i ngā mea poke, me whai i te iti rawa o te 10 meneti te roa o te whakapā atu.
Heoi anō, ka taea te whakamahi i ngā hua arumoni kua whakamanahia hei kaiwhakakore poke mata whakangaro DNA mēnā kāore e whakaaetia te whakamahinga o te konutai haukōroiti i roto i ngā taunakitanga haumaru ā-rohe, mēnā rānei kāore e pai te konutai haukōroiti mō te whakakore poke i ngā wāhanga whakarewa o ngā taputapu.
Ko te mea pai, me whai ngā kaimahi i te kaupapa rere mahi kotahi-ara, kaua hoki e hoki atu i ngā wāhi paru (whai muri i te PCR) ki ngā wāhi ma (i mua i te PCR) i taua rā anō. Heoi anō, tera pea ētahi wā kāore e taea te karo i tēnei. Mēnā ka puta mai tēnei āhuatanga, me āta horoi ngā kaimahi i ō rātou ringaringa, me whakawhiti karapu, me whakamahi i te koti taiwhanga kua tohua, ā, kaua hoki e whakauru mai i ngā taputapu e hiahia ana rātou ki te tango atu i waho o te rūma, pērā i ngā pukapuka taiwhanga. Me whakanui ēnei tikanga whakahaere i roto i ngā whakangungu kaimahi mō ngā tikanga ngota.
I muri i te whakamahinga, me horoi ngā wāhi noho ki te 10% konutai hauhāoraiti (ā, muri iho me te wai parakore hei tango i te wai mākū toenga), te 70% etanoru, te wai whakamāori rānei kua whakamanahia hei whakangaro i te DNA e wātea ana i te mākete. Ko te mea pai, me whakauru ngā rama hihi-paura (UV) hei whakahohe i te whakamāoritanga mā te hihi. Heoi, me whakawhāiti te whakamahinga o ngā rama UV ki ngā wāhi mahi kua kati, hei tauira, ki ngā kāpata haumaru, kia kore ai e pāngia ngā kaimahi taiwhanga e te hihi UV. Me whai i ngā tohutohu a te kaihanga mō te tiaki i ngā rama UV, te whakapūhautanga me te horoi kia mau tonu ai te whai hua o ngā rama.
Mena kei te whakamahia te 70% etanoru hei utu mō te konutai haikoroiti, me whiti te rama UV hei whakaoti i te whakakorenga.
Kaua e horoia te vortex me te centrifuge ki te sodium hypochlorite; engari, horoia ki te 70% ethanol ka waiho ki te mārama UV, whakamahia rānei he decontamination whakangaro DNA arumoni. Mō ngā maringi, whakapā atu ki te kaihanga mō ētahi atu tohutohu horoi. Mena ka whakaaetia e ngā tohutohu a te kaihanga, me whakahoromata tonu ngā pipette mā te autoclave. Mena kāore e taea te autoclave i ngā pipette, me horoi noa ki te 10% sodium hypochlorite (ā muri iho ka murua pai ki te wai parakore) me te decontamination whakangaro DNA arumoni rānei, ā muri iho ka whakatōngia ki te UV.
Mā te horoi ki te konutai hauhā nui ka kino pea ngā kirihou me ngā konganuku o te pipette mēnā ka mahia i ia wā; tirohia ngā taunakitanga a te kaihanga i te tuatahi. Me whakatikatika auau ngā taputapu katoa kia rite ki te kaupapa i tūtohutia e te kaihanga. Me whai kawenga tētahi tangata kua tohua hei whakarite kia piri ki te kaupapa whakatikatika, kia mau tonu ngā rārangi taipitopito, ā, kia mārama hoki te whakaatu i ngā tapanga ratonga ki ngā taputapu.
3. Ngā tohutohu mō te whakamahinga me te horoi mō te wāhi ngota ngota kua tohua
Pre-PCR: Te whakarite i te whakawehewehenga matū/te whakarite i te ranunga matua: Ko tēnei te wāhi tino ma o ngā wāhi katoa e whakamahia ana mō te whakarite i ngā whakamātautau ngota, ā, ko te mea pai ko tētahi kāpata rere laminar kua tohua me te rama UV. Kaua ngā tauira, te waikawa karihi kua tangohia me ngā hua PCR kua whakanuia e whakahaerea ki tēnei wāhi. Me pupuri ngā matū whakanui i roto i te pouaka tio (i roto rānei i te pouaka whakamātao, e ai ki ngā taunakitanga a te kaihanga) i te wāhi kotahi kua tohua, ko te mea pai ko te taha o te kāpata rere laminar, o te wāhi pre-PCR rānei. Me whakawhiti ngā karapu i ia wā e tomo ana ki te wāhi pre-PCR, ki te kāpata rere laminar rānei.
Me horoi te wāhi i mua i te PCR, te kāpata rere laminar rānei i mua, i muri hoki i te whakamahinga penei: Horoia ngā mea katoa i roto i te kāpata, hei tauira, ngā pipette, ngā pouaka pito, te vortex, te centrifuge, ngā whata ngongo, ngā pene, me ētahi atu ki te 70% etanoru, ki tētahi mea horoi poke whakangaro DNA arumoni rānei, ā, waiho kia maroke. Mēnā he wāhi mahi kua kati, hei tauira, he kāpata rere laminar, whakaaturia te taupoki ki te mārama UV mō te 30 meneti.
Tuhipoka
Kaua e whakaaturia ngā matū matū ki te mārama UV; me neke noa ki roto i te kāpata ina kua ma. Mena kei te whakahaerehia te PCR whakamuri, he pai pea te muru i ngā mata me ngā taputapu ki tētahi otinga e wawahi ana i ngā RNase ina pā atu. Mā tēnei pea ka āwhina ki te karo i ngā hua kino teka mai i te pakaru o te whākōkī o te RNA. Whai muri i te horoinga poke, ā, i mua i te whakarite i te mastermix, me whakawhiti anō ngā karapu, kātahi ka rite te kāpata ki te whakamahi.
Pre-PCR: Tangohanga waikawa karihi/tāpiri tauira:
Me tango te waikawa karihi, ā, me whakahaere ki tētahi atu wāhi kua tohua, mā te whakamahi i tētahi huinga motuhake o ngā pipettes, ngā pito tātari, ngā whata ngongo, ngā karapu hou, ngā koti taiwhanga me ētahi atu taputapu. Kei tēnei wāhi anō hoki te tāpiri i ngā tauira, ngā mana whakahaere me ngā rārangi au ki ngā ngongo, ki ngā pereti rānei o te mastermix. Hei karo i te poke o ngā tauira waikawa karihi kua tangohia e tātarihia ana, e taunakihia ana kia whakawhiti karapu i mua i te whakahaere i ngā mana whakahaere pai, i ngā paerewa rānei, me te whakamahi i tētahi huinga motuhake o ngā pipettes. Kaua ngā matū PCR me ngā hua kua whakanuia e tukuna ki tēnei wāhi. Me rongoa ngā tauira ki roto i ngā pouaka whakamātao, ki ngā pouaka tio rānei kua tohua ki te wāhi kotahi. Me horoi te wāhi mahi tauira i te ara kotahi me te wāhi mastermix.
Muri i te PCR: Te whakanui me te whakahaere i te hua kua whakanuia
Ko tēnei wāhi kua tohua mō ngā tukanga whai muri i te whakanui, ā, me wehe kē atu i ngā wāhi o mua i te PCR. Kei roto i te nuinga o te wā ngā mīhini whakamahana me ngā tūāpapa wā-tūturu, ā, ko te mea pai me whai kāpata rere laminar hei tāpiri i te hua PCR rauna 1 ki te tauhohenga rauna 2, mēnā kei te whakahaerehia te PCR kōpiko. Kaua e whakahaerea ngā matū PCR me te waikawa karihi kua tangohia ki tēnei wāhi nā te mea he nui te tūponotanga o te poke. Me whai motuhake tēnei wāhi i ngā karapu, ngā koti taiwhanga, ngā whata pereti me ngā ngongo, ngā paipa, ngā pito tātari, ngā ipu me ētahi atu taputapu. Me whakapūtā ngā ngongo i mua i te whakatuwheratanga. Me horoi te wāhi mahi tauira i te ara kotahi me te wāhi mastermix.
I muri i te PCR: Te tātaritanga hua
Ko tēnei rūma he wāhi mō ngā taputapu kimi hua, hei tauira, ngā tāke hikohiko gel, ngā kete hiko, te rama whakawhiti UV me te pūnaha tuhinga gel. Me whai huinga karapu motuhake tēnei wāhi, ngā koti taiwhanga, ngā whata pereti me ngā ngongo, ngā paipa, ngā pito tātari, ngā ipu me ētahi atu taputapu. Kāore e whakaaetia kia kawea mai ētahi atu matū tauhohe ki tēnei wāhi, haunga te tae uta, te tohu ngota me te agarose gel, me ngā wāhanga ārai. Me horoi te wāhi mahi tauira i te ara kotahi me te wāhi mastermix.
He kōrero nui
Ko te mea pai, kaua e tomo ki ngā rūma pre-PCR i te rā kotahi mēnā kua oti kē te mahi i roto i ngā rūma post-PCR. Mēnā he mea tino kore e taea te karo, me whakarite kia horoia pai ō ringaringa i te tuatahi, ā, me mau hoki he koti taiwhanga motuhake ki roto i ngā rūma. Kaua e haria ngā pukapuka taiwhanga me ngā pepa ki roto i ngā rūma pre-PCR mēnā kua whakamahia i roto i ngā rūma post-PCR; mēnā e tika ana, tangohia he tānga tārua o ngā kawa/tauira ID, me ētahi atu.
4. Ngā tohutohu whānui mō te koiora ngota
Whakamahia he karapu kore-paura hei karo i te aukati i te whakamātautau. He mea nui te tikanga tika o te whakapūpū paipa hei whakaiti i te poke. Mā te whakapūpū paipa ka tauhiuhi te wai i te wā e tukuna ana, ā, ka puta he haunga. Ka kitea ngā mahi pai mō te whakapūpū paipa i ngā hononga e whai ake nei: Gilson guide to pipetting, Anachem pipetting technique videos, Ngā ngongo centrifuge i mua i te whakatuwheratanga, ā, whakatuwheratia kia tūpato kia kore ai e tauhiuhi. Katia tonutia ngā ngongo i muri i te whakamahinga hei karo i te urunga mai o ngā poke.
I te wā e mahi ana i ngā tauhohenga maha, whakaritea tētahi ranunga matua kei roto ngā tauhohenga noa (hei tauira, te wai, ngā dNTP, te pūhui, ngā paraime me te whākōkī) hei whakaiti i te maha o ngā whakawhiti tauhohenga me te whakaiti i te tūponotanga o te poke. E taunakihia ana kia whakatūria te ranunga matua ki runga i te hukapapa, ki runga rānei i tētahi poraka matao. Mā te whakamahi i tētahi whākōkī Tīmatanga Wera ka āwhina pea ki te whakaiti i te whakaputanga o ngā hua kore-motuhake. Tiakina ngā tauhohenga kei roto ngā pūwero kanapa mai i te mārama kia kore ai e pirau.
5. Ngā mana whakahaere ā-roto
Whakaurua ngā mana whakahaere pai me ngā mana whakahaere kino kua tautuhia pai, kua whakaūtia, me te mana whakahaere kore-tauira i roto i ngā tauhohenga katoa, me te rārangi ia ine maha-ira mō ngā tauhohenga tauanga. Kaua te mana whakahaere pai e tino kaha kia tūpono ai te poke. Whakaurua ngā mana whakahaere tango pai me kino i te wā e mahi ana i te tango waikawa karihi.
E taunakihia ana kia whakairihia ngā tohutohu mārama ki ia wāhi kia mōhio ai ngā kaiwhakamahi ki ngā ture whanonga. Ko ngā taiwhanga tātaritanga e kite ana i ngā taumata tino iti o te DNA, o te RNA rānei i roto i ngā tauira haumanu, me whakamahi pea i te tikanga haumaru tāpiri mā te whai pūnaha whakahaere hau motuhake me te pēhanga hau paku pai i roto i ngā rūma i mua i te PCR, me te pēhanga hau paku kino i roto i ngā rūma i muri i te PCR.
Hei whakamutunga, he mea āwhina te whakawhanake i tētahi mahere whakapūmau kounga (QA). Me whakauru ki roto i tēnei mahere ngā rārangi o ngā taonga matua o ngā matū matū me ngā taonga mahi, ngā ture mō te penapena i ngā kete me ngā matū matū matū, te pūrongo i ngā hua whakahaere, ngā kaupapa whakangungu kaimahi, ngā rauropi rapurongoā, me ngā mahi whakatika ina hiahiatia.
6. Rārangi Pukapuka
Aslan A, Kinzelman J, Dreelin E, Anan'eva T, Lavander J. Upoko 3: Te whakatū i tētahi taiwhanga qPCR. He tuhinga aratohu mō te whakamātautau i ngā wai whakangahau mā te whakamahi i te tikanga USEPA qPCR 1611. Lansing- Te Whare Wānanga o te Kāwanatanga o Michigan.
(Te Hauora Tūmatanui o Ingarangi, NHS. Ngā paerewa o te UK mō ngā rangahau moroiti: Te Mahi Taiwhanga Pai i te wā e mahi ana i ngā whakamātautau whakanui ngota). Aratohu Kounga. 2013;4(4):1–15.
Mifflin T. Te whakatū i tētahi taiwhanga PCR. Cold Spring Harb Protoc. 2007;7.
Schroeder S 2013. Te tiaki auau o ngā centrifuge: te horoi, te tiaki me te patu huakita o ngā centrifuge, ngā rotors me ngā adapters (Pepa Mā Nama 14). Hamburg: Eppendorf; 2013.
Viana RV, Wallis CL. Te Mahi Taiwhanga Hauora Pai (GCLP) mō ngā whakamātautau irahiko e whakamahia ana i roto i ngā taiwhanga tātaritanga, I roto i: Akyar I, ētita. Ngā whānuitanga o te mana whakahaere kounga. Rijeka, Croatia: Intech; 2011: 29–52.
Wā tuku: Hōngongoi-16-2020
