I-PCR Analyser Troubleshooting: Imibuzo edla ngokubuzwa kunye nezisombululo

I-Polymerase chain reaction (PCR) abahlalutyi zizixhobo ezibalulekileyo kwibhayoloji ye-molekyuli, evumela abaphandi ukuba bakhulise i-DNA yezicelo ezisuka kuphando lwemfuzo ukuya kuxilongo lweklinikhi. Nangona kunjalo, njengaso nasiphi na isixhobo esiyinkimbinkimbi, i-PCR analyzer inokuhlangabezana neengxaki ezichaphazela ukusebenza kwayo. Eli nqaku liphendula imibuzo eqhelekileyo malungaPCR analyzerukulungisa iingxaki kunye nokubonelela ngezisombululo ezisebenzayo kwiingxaki eziqhelekileyo.

1. Kutheni isenzo sam sePCR singandisi?

Enye yeengxaki eziqhelekileyo abajongene nazo abasebenzisi kukungakwazi kwe-PCR reaction ukukhulisa i-DNA ekujoliswe kuyo. Oku kunokubalelwa kwizinto ezininzi:

Uyilo lwe-primer olungalunganga: Qinisekisa ukuba ii-primers zakho zichanekileyo ngokulandelelana okujoliswe kuyo kwaye zibe nobushushu obuphezulu bokunyibilika (Tm). Sebenzisa izixhobo zesoftware kuyilo lweprimer ukunqanda ukubophelela okungachazwanga.

Ayonelanga Template DNA: Qinisekisa ukuba usebenzisa isixa esaneleyo setemplate yeDNA. Okuncinci kakhulu kuya kubangela ukuba buthathaka okanye kungabikho ukukhulisa.

I-inhibitors kwisampuli: Ukungcola kwisampuli kunokuthintela ukusabela kwe-PCR. Cinga ngokucoca i-DNA yakho okanye usebenzise indlela eyahlukileyo yokukhupha.

Isisombululo: Jonga uyilo lwakho lwe-primer, ukwandisa ukugxininiswa kwetemplate, kwaye uqinisekise ukuba isampuli yakho ayinayo inhibitors.

2. Kutheni imveliso yam ye-PCR inobungakanani obungalunganga?

Ukuba ubungakanani bemveliso yakho yePCR abukho njengoko kulindelekile, bunokubonisa ingxaki ngeemeko zokusabela okanye izithako ezisetyenzisiweyo.

Ukwandisa okungangqalanga: Oku kunokwenzeka ukuba i-primer ibophelela kwisiza esingacetywanga. Jonga izinto ezicacileyo zeprimers usebenzisa isixhobo esifana ne-BLAST.

Ubushushu be-Annealing obungachanekanga: Ukuba iqondo lobushushu le-annealing lisezantsi kakhulu, kungavela ukubophelela okungangqalenga. Ukuphucula ubushushu bokucoca nge-PCR yegradient.

Isisombululo: Qinisekisa ukuchaneka kwe-primer kunye nokwandisa ubushushu be-annealing ukuphucula ukuchaneka kweemveliso ze-PCR.

3. Uhlalutyi lwePCR yam lubonisa umyalezo wemposiso. Ndingenza ntoni?

Imiyalezo yemposiso kwi-PCR analyzer inokothusa, kodwa ihlala ibonelela ngemikhondo kwiingxaki ezinokubakho.

Imiba yokulinganisa: Qinisekisa ukuba i-PCR analyzer ilinganiswe ngokuchanekileyo. Ukugcinwa rhoqo kunye nokuhlolwa kokulungelelaniswa kubalulekile ekufumaneni iziphumo ezichanekileyo.

Iqela leSoftware: Ngamanye amaxesha, iibugs zesoftware zinokubangela iingxaki. Qala kwakhona ikhompyuter yakho kwaye ujonge uhlaziyo lwesoftware.

ISISOMBULULO: Jonga kwincwadana yomsebenzisi yekhowudi ethile yempazamo kwaye ulandele amanyathelo acetyiswayo okusombulula ingxaki. Ukugcinwa rhoqo kunokuthintela iingxaki ezininzi.

4. Kutheni iziphumo zempendulo ye-PCR yam zingangqinelani?

Iziphumo ezingahambelaniyo zePCR zinokudanisa ngenxa yezizathu ezininzi:

Umgangatho we-Reagent: Qinisekisa ukuba zonke ii-reagents, kubandakanywa ii-enzymes, ii-buffers, kunye ne-dNTPs, zintsha kwaye zikumgangatho ophezulu. Ii-reagents eziphelelwe lixesha okanye ezingcolisekileyo zinokubangela ukuguquguquka.

I-Thermal Cycler Calibration: Imimiselo yokushisa engahambelaniyo inokuchaphazela inkqubo ye-PCR. Jonga rhoqo ukulinganisa kwe-thermal cycler.

Isisombululo: Sebenzisa ii-reagents ezikumgangatho ophezulu kwaye ulungelelanise ibhayisikili yakho ye-thermal rhoqo ukuze uqinisekise iziphumo ezihambelanayo.

5. Indlela yokuphucula ukusebenza kakuhle kwe-PCR reaction?

Ukuphucula ukusebenza kakuhle kwe-PCR reactions kunokukhokelela kwizivuno eziphezulu kunye neziphumo ezithembekileyo.

Lungiselela iimeko zokusabela: Linga usebenzisa ugxininiso olwahlukeneyo lweeprimers, itemplate yeDNA kunye neMgCl2. Indlela nganye ye-PCR inokufuna iimeko ezizodwa ukuze isebenze ngokugqibeleleyo.

Sebenzisa i-enzymes ephezulu yokuthembeka: Ukuba ukuchaneka kubalulekile, cinga ukusebenzisa i-polymerase ye-DNA ephezulu yokuthembeka ukunciphisa iimpazamo ngexesha lokukhulisa.

Isisombululo: Yenza umfuniselo wokuphucula ukufumana ezona meko zilungileyo zokuseta iPCR yakho.

Isishwankathelo

Ukusombulula ingxaki aPCR analyzerinokuba ngumsebenzi onzima, kodwa ukuqonda iingxaki eziqhelekileyo kunye nezisombululo zazo kunokwandisa kakhulu amava akho ePCR. Ngokusombulula ezi ngxaki eziqhelekileyo, abaphandi banokuphucula iziphumo ze-PCR kwaye baqinisekise iziphumo ezithembekileyo kwizicelo zebhayoloji ye-molekyuli. Ukugcinwa rhoqo, ukukhethwa ngononophelo kwee-reagents, kunye nokulungiswa kweemeko zokusabela zizitshixo kuhlalutyo oluyimpumelelo lwe-PCR.