Senza nokuhlolwa kwe-PCR, kungani imiphumela ihlale imibi kakhulu kuneminye?

Senza nokuhlolwa kwe-PCR, kungani imiphumela ihlale imibi kakhulu kuneminye?

Elabhorethri, bheka i-PCR yabanye abantu, ukusebenza kahle kwe-amplification kuphezulu, i-electrophoresis strip iphelele, futhi ubheke ezabo, okunzima ukuzenza isikhathi eside, uzwe ukusabela kukuhle kakhulu, umphumela wokuhlolwa okungamanga …… inkinga?

Ngikholwa ukuthi abaningi esibambisene nabo bahlangabezane nezinkinga ezahlukene.Uma singena odongeni ekuhlolweni kwe-PCR, sizohlaziya izici ezingaphakathi kwesistimu yokusabela, kodwa ngokuvamile sishaya indiva ithonya elibalulekile lezinto ezisebenzisekayo ekuhloleni.

Umphumela wakho we-PCR awulungile, okusebenzisekayo kwe-PCR kungase kube isizathu esibalulekile.Ngakolunye uhlangothi, ukungcoliswa okuncane kwezinto ezisetshenziswayo noma ukwethulwa kwama-inhibitors kuzodala ukungcola kokuhlolwa;Ngakolunye uhlangothi, ukukhetha okungafanele kwezinto ezisetshenziswayo nakho kuzokwenza imiphumela yokuhlola ihlupheke.

Inkinga ibuya futhi.Kunezinhlobo eziningi ze-PCR ezisebenzisekayo, ngakho-ke ungakhetha kanjani ezifanele ukuze wenze isilingo sibe bushelelezi?Yiziphi izinkinga okufanele zinakwe?Ungakhathazeki, namuhlaI kuhlangene neminyaka eminingi yokuhlangenwe nakho, ukuxazulula izinkinga ezivamile zokukhetha izinto ezisetshenziswayo ze-PCR, ngithemba ukukusiza.

 

Q1: Kungani izinto ezisetshenziswayo ze-PCR ngokuvamile zenziwe ngezinto ze-PP?

I-Huida: Ngenxa yokuthi ukunikezwa kwe-PCR kuvame ukuthintana ngokuqondile nama-reagents noma amasampula, izinto ze-polypropylene (PP) azifaki ngokwebhayoloji, azinamatheli kuma-biomoleculars, futhi zinokubekezelela okuhle kwamakhemikhali nokubekezelelana kwezinga lokushisa (zingashaywa ngokuzenzakalelayo ku-121° C, ingamelana nokushintsha kwezinga lokushisa phakathi nomjikelezo wokushisa).

 

Q2: Kufanele ngikhethe kanjani phakathi kosayizi abahlukene bamashubhu/amapuleti e-PCR?

I-HUIDA: Inhloso yokukhetha: khetha imikhiqizo efanele ngokuya ngezidingo ezithile zokuhlola.Iningi losayizi weshubhu le-PCR lingahlangabezana nezidingo zokusabela kwe-PCR.Ngalesi sisekelo, kunconywa ukuthi ukhethe ishubhu yevolumu ephansi kuqala.Ngenxa yokuthi ishubhu/ipuleti lokusabela kwevolumu ephansi linendawo encane engaphezulu, ukuqhutshwa kokushisa kuyathuthukiswa futhi ukuhwamuka kuyancishiswa.Ngesikhathi esifanayo, kuyadingeka ukugwema ukungeza amasampula amaningi kakhulu noma amancane kakhulu.Ukuningi kakhulu kungaholela ekwehleni kokuhanjiswa kwe-thermal, ukuchitheka nokungcoliswa kwesiphambano, kuyilapho kuncane kakhulu kungase kuphumele ekulahlekeni kokuhwamuka kwesampula.

Imininingwane namavolumu amashubhu okuphendula ajwayelekile:

Ishubhu eyodwa/ehlanganisiwe: 0.5ml, 0.2ml, 0.15ml

Ipuleti lemithombo engu-96: 0.2ml, 0.15ml

Ipuleti lemithombo engu-384: 0.04ml

 

Q3: Uma inani lesampula lilincane, ngizokhetha ipayipi elilodwa noma ipayipi elihlanganisiwe, kodwa kungani amanye enekhava eyisicaba kanti amanye anesembozo seconvex?

I-Huida: Uma inani lesampula lincane, ithubhu eyodwa noma ishubhu ehlanganisiwe izokhethwa.Kodwa-ke, lapho umthamo wokusabela mkhulu, ishubhu elilodwa linamandla, futhi umthamo ungafinyelela ku-0.5 mL.Futhi ikhava flat kanye convex ikhava ngamunye has inzuzo, ethize angabhekisela ulwazi olulandelayo.

I-tube eyodwa: 0.2ml no-0.5ml;Ingakhetha ubuningi besampula ngendlela evumelana nezimo.

Ithubhu yokuxhuma: i-0.2ml noma i-0.15ml iyakhethwa;Amashubhu angu-8 noma angu-12 avamile.

Ikhava eyisicaba: ingahlinzeka ngokudluliswa kwesignali ye-fluorescence enembile ye-qPCR;Kulula ukubhala amamaki.

Isembozo se-convex: ukuthintana nesembozo esishisayo sensimbi ye-PCR ukuze unciphise ukuwohloka kweshubhu lokusabela okubangelwa ingcindezi;Nokho, izothinta ukudluliswa kwesignali ye-fluorescence futhi ayikwazi ukusetshenziswa ekuhlolweni kwe-qPCR.

 

Q4: Kungani amanye amapuleti e-PCR eneziketi kanti amanye angenazo?

I-Huida: Eqinisweni, isiketi sepuleti ye-PCR senzelwe ukuzivumelanisa kangcono nezinhlelo zokusebenza ezizenzakalelayo, ukuhlinzeka ngokusekela okuzinzile nokukhuthazela kwemishini kwensimbi, kanye nokuzinza okuphezulu phakathi nenqubo yepayipi.

Amapuleti e-PCR ngokuvamile ahlukaniswa abe i-non-hemline, i-half-hemline kanye ne-hemline egcwele.

NgaphandleIpuleti: Ifanele imishini eminingi ye-PCR noma imishini ye-qPCR, kodwa hhayi izinhlelo zokusebenza ezizenzakalelayo.Ukuzinza akuphakeme ohlelweni lwe-pipetting, ngakho-ke kudinga ukusetshenziswa ngokusekelwa kwepuleti.

Ipuleti lomphetho wesiketi esiyingxenye: ingashintshwa ukuze ilebula noma isicelo sekhodi yebha, kanye nokusetshenziswa okuzenzakalelayo, futhi inokuqina okuhle kwamapayipi.

Ipuleti lonqenqema lwesiketi esigcwele: lilungele kakhulu izinhlelo zokusebenza zokuhlola ezizenzakalelayo, lingashintshwa futhi libe ilebula nokusetshenziswa kwekhodi yebha.Inamandla amahle emishini, ingasetshenziswa ku-bulge module PCR instrument, futhi inozinzo oluphezulu ohlelweni lwe-pipetting.

 

Q5 :Kumapuleti e-PCR, kungani amakhona namalebula engafani nhlobo?

I-Huida: Kuncike emphumeleni wokusika ekhoneni kanye nokumaka.

Ukusika amakhona: Ukukhetha indawo yokusika ekhoneni lepuleti le-PCR kuncike ezidingweni zethuluzi elifanele lokubeka indawo kalula.

Ukuhlonza: Omaka be-alphanumeric kumapuleti e-PCR basiza ukukhomba Imithombo ngayinye nezindawo zesampula.Ngokuvamile kuphawu lwedijithali lombala oqoshiwe noma ilogo eqoshiwe.Kwezinye izinhlelo zokusebenza ezizenzakalelayo, amapuleti okusabela aphrintiwe namakiwe avalwa kangcono.

 

Q6: Kungani amanye amapuleti e-PCR e-convex kuyilapho amanye eyisicaba?Yikuphi okungcono?

U-Huida: Okufanelana kuhle kakhulu.Kunezinhlobo ezimbili zamapuleti avamile e-PCR: i-pore margin eyisicaba kanye ne-high pore margin.

Ipuleti yokusabela ye-flat hole margin, ilungele izisetshenziswa eziningi ze-PCR.

Amapuleti okusabela anemiphetho ekhuphukayo enza kube lula ukuvala ulwelwesi futhi anciphise ubungozi bokungcola phakathi kwamasampuli.

 

U-Huida uneminyaka eminingi yokuhlangenwe nakho ekukhiqizeni nasekusetshenzisweni kwemikhiqizo ye-PCR kanye namazinga aphezulu embonini.Ulayini womkhiqizo unothile futhi ungahlangabezana nenani elikhulu lezinhlelo zokusebenza zokuhlola ze-PCR.Woza ukhethe imikhiqizo efanele yezinhlelo zakho zokusebenza zokuhlola.

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Isikhathi sokuthumela: Jun-03-2021

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