I-PCR Analyser Troubleshooting: Imibuzo Evame Ukubuzwa Nezisombululo

Abahlaziyi be-Polymerase chain reaction (PCR) bangamathuluzi abalulekile kubhayoloji yamangqamuzana, okuvumela abacwaningi ukuthi bakhulise i-DNA yezinhlelo zokusebenza ezisukela ocwaningweni lofuzo kuye ekuxilongweni komtholampilo. Nokho, njenganoma iyiphi idivayisi eyinkimbinkimbi, i-PCR analyzer ingahlangabezana nezinkinga ezithinta ukusebenza kwayo. Lesi sihloko sikhuluma ngemibuzo evamile mayelanaPCR analyzerukuxazulula izinkinga futhi inikeza izixazululo ezingokoqobo ezinkingeni ezivamile.

1. Kungani ukusabela kwami ​​kwe-PCR kungakhulisi?

Enye yezinkinga ezivame kakhulu abasebenzisi ababhekana nazo ukungakwazi ukusabela kwe-PCR ukukhulisa i-DNA eqondiwe. Lokhu kungabangelwa izici ezimbalwa:

Idizayini ye-primer engalungile: Qiniseka ukuthi ama-primers akho aqondile ngokulandelana okuqondiwe futhi anezinga lokushisa elifanele lokuncibilika (Tm). Sebenzisa amathuluzi esofthiwe edizayini yokuqala ukuze ugweme ukubopha okungaqondile.

I-DNA Yesifanekiso Enganele: Qinisekisa ukuthi usebenzisa inani elanele lesifanekiso se-DNA. Ukuncane kakhulu kuzoholela ekukhulisweni okubuthakathaka noma okungekho.

Ama-inhibitors kusampula: Ukungcola kusampula kungavimbela ukusabela kwe-PCR. Cabangela ukuhlanza i-DNA yakho noma usebenzise indlela ehlukile yokukhipha.

Isixazululo: Hlola ukwakheka kwakho kwe-primer, khulisa ukugxilisa kwesifanekiso, futhi uqiniseke ukuthi isampula yakho ayinawo ama-inhibitors.

2. Kungani umkhiqizo wami we-PCR ungusayizi ongalungile?

Uma usayizi womkhiqizo wakho we-PCR ungekho njengoba bekulindelekile, kungase kubonise inkinga ngezimo zokusabela noma izithako ezisetshenzisiwe.

Ukukhulisa okungaqondile: Lokhu kungenzeka uma i-primer ibophezela kusayithi elingahlosiwe. Hlola ukucaciswa kweziqalisi usebenzisa ithuluzi elifana ne-BLAST.

I-Annealing Temperature Engalungile: Uma izinga lokushisa le-anneal liphansi kakhulu, kungase kubangele ukubophezela okungaqondile. Ukuthuthukiswa kwezinga lokushisa le-annealing nge-gradient PCR.

Isixazululo: Qinisekisa ukucaciswa kwe-primer futhi wandise izinga lokushisa lokudonsa ukuze uthuthukise ukunemba kwemikhiqizo ye-PCR.

3. Isihlaziyi se-PCR yami sibonisa umyalezo wephutha. kumele ngenzeni?

Imilayezo yephutha ekuhlaziyweni kwe-PCR ingethusa, kodwa ngokuvamile inganikeza imikhondo ezinkingeni ezingaba khona.

Izinkinga Zokulinganisa: Qinisekisa ukuthi isihlaziyi se-PCR silinganiswe ngendlela efanele. Ukugcinwa okuvamile nokuhlolwa kokulinganisa kubalulekile ukuze uthole imiphumela enembile.

Iqembu Lesoftware: Kwesinye isikhathi, iziphazamisi zesoftware zingadala izinkinga. Qala kabusha ikhompuyutha yakho futhi uhlole izibuyekezo zesofthiwe.

ISIXAZULULO: Bheka imanuwali yomsebenzisi ngekhodi ethile yephutha bese ulandela izinyathelo ezinconyiwe zokuxazulula inkinga. Ukugcinwa njalo kungavimbela izinkinga eziningi.

4. Kungani imiphumela yokusabela kwe-PCR yami ingahambisani?

Imiphumela ye-PCR engahambisani ingase ikhungathekise ngenxa yezizathu ezimbalwa:

Ikhwalithi ye-Reagent: Qinisekisa ukuthi zonke izinto ezivuselelayo, okuhlanganisa ama-enzyme, amabhafa, nama-dNTP, masha futhi asezingeni eliphezulu. Ama-reagents aphelelwe yisikhathi noma angcolile angabangela ukuhlukahluka.

I-Thermal Cycler Calibration: Izilungiselelo zokushisa ezingahambisani zingathinta inqubo ye-PCR. Hlola njalo ukulinganiswa komjikelezo oshisayo.

Isixazululo: Sebenzisa ama-reagents ekhwalithi ephezulu futhi ulinganise ibhayisikili lakho elishisayo njalo ukuze uqinisekise imiphumela engaguquki.

5. Ungathuthukisa kanjani ukusebenza kahle kwe-PCR?

Ukuthuthukisa ukusebenza kahle kokusabela kwe-PCR kungaholela ekuvuneni okuphezulu kanye nemiphumela ethembeke kakhulu.

Lungiselela izimo zokusabela: Linga usebenzisa ukugxilisa okuhlukene kweziqalo, isifanekiso se-DNA ne-MgCl2. Ukusabela ngakunye kwe-PCR kungase kudinge izimo ezihlukile ukuze kusebenze kahle.

Sebenzisa ama-enzyme athembekile kakhulu: Uma ukunemba kubalulekile, cabanga ukusebenzisa i-polymerase ye-DNA ethembekile ukuze unciphise amaphutha ngesikhathi sokukhulisa.

Isixazululo: Yenza isilingo sokuthuthukisa ukuze uthole izimo ezihamba phambili zokusetha i-PCR yakho ethile.

Ngokufigqiwe

Ukuxazulula inkinga aPCR analyzerkungaba umsebenzi onzima, kodwa ukuqonda izinkinga ezivamile nezisombululo zazo kungathuthukisa kakhulu ulwazi lwakho lwe-PCR. Ngokuxazulula lezi zinkinga ezivamile, abacwaningi bangathuthukisa imiphumela ye-PCR futhi baqinisekise imiphumela enokwethenjelwa ekusetshenzisweni kwebhayoloji yamangqamuzana. Ukunakekelwa okuvamile, ukukhethwa ngokucophelela kwama-reagents, kanye nokwenza kahle kwezimo zokusabela kuyizihluthulelo zokuhlaziya okuyimpumelelo kwe-PCR.