I-Robustart Taq DNA Polymerase

I-Robustart Taq DNA Polymerase iyisiqalo esishisayo se-DNA polymerase.Lo mkhiqizo awukwazi ukuvimbela kangcono kuphela ukusabela okungaqondile okubangelwa ukuhunyushwa okungaqondile kwama-primers noma ukuhlanganiswa kwe-primer kunqubo yokulungiselela nokukhulisa uhlelo lwe-PCR.Ngakho-ke, inokucaciswa okuhle kakhulu futhi isebenza kangcono ekwandiseni izifanekiso zokugxilisa ingqondo eziphansi, futhi ifanele ukusabela kokukhulisa i-PCR okuphindaphindiwe.Ngaphezu kwalokho, lo mkhiqizo unokusebenziseka okuhle kakhulu, futhi imiphumela yokukhulisa izinzile ingatholwa ngezinhlobo ezahlukene zokusabela kwe-PCR.

Izingxenye

1.5 U/μL I-Robustart Taq DNA polymerase

2.10 × PCR Buffer II (Mg²+ mahhala) (uma uthanda)

3.25 mM MgCl₂(uyazikhethela)

* I-10 × PCR Buffer II (Mg²+ mahhala) ayiqukethe i-dNTP ne-Mg²+, sicela wengeze i-dNTP ne-MgCl₂lapho ulungiselela uhlelo lokusabela.

Izicelo Ezinconyiwe

1.Ukukhulisa ngokushesha.

2.Ukukhulisa okuningi.

3.Ukukhuliswa okuqondile kwegazi, ama-swabs, namanye amasampula.

4.Ukutholwa kwezifo zokuphefumula.

Isimo Sesitoreji

-20°C ukuze igcinwe isikhathi eside, kufanele ixutshwe kahle ngaphambi kokusetshenziswa, gwema ukuncibilika kweqhwa njalo.

*Uma imvula iba ngemva kwesiqandisi, kujwayelekile;kunconywa ukulinganisa izinga lokushisa ekamelweni ngaphambi kokuxuba nokusebenzisa.

Incazelo Yeyunithi

Iyunithi eyodwa esebenzayo (U) ichazwa njengenani le-enzyme elihlanganisa i-10 nmol ye-deoxyribonucleotide kwinto engancibiliki i-asidi ku-74°C imizuzu engu-30 kusetshenziswa i-DNA yesidoda se-salmon ecushiwe njengesifanekiso/i-primer.

Ikhwalithi yokulawula

1.Ukuhlanzeka kwe-electrophoretic ye-SDS-PAGE okukhulu kuno-98%.

2.Ukuzwela kokukhulisa, ukulawula kwe-batch-to-batch, ukuzinza.

3.Awukho umsebenzi we-nuclease exogenous, akukho endonuclease exogenous noma exonuclease ukungcola

Iziyalezo

Ukusethwa kokusabela

Amanothi:

1) a.I-buffer ayiqukethe i-dNTP ne-Mg²+, sicela wengeze i-dNTPs ne-MgCl₂lapho ulungiselela uhlelo lokusabela.

2) b.Uma isetshenziselwa i-qPCR/qRT-PCR, ama-fluorescent probe kufanele engezwe ohlelweni lokusabela.Ngokuvamile, ukuhlushwa kwe-primer yokugcina ye-0.2 μM kuzonikeza imiphumela emihle;uma ukusebenza kokusabela kukubi, ukugxilwa kwe-primer kungalungiswa ebangeni lika-0.2-1 μM.Ukugxila kwe-probe kuvame ukwenziwa ngcono ku-0.1-0.3 μM.Ukuhlolwa kwe-concentration gradient kungenziwa ukuze kutholwe inhlanganisela engcono kakhulu ye-primer ne-probe.

Iphrothokholi yokuhamba ngebhayisikili eshisayo

Amanothi

1.Izinga lokukhulisa i-DNA polymerase esheshayo akufanele libe ngaphansi kuka-1 kb/10 s.Izinga lokushisa lokunyuka nokuwa, imodi yokulawula izinga lokushisa nokusebenza kahle kokwenziwa kokushisa kwamathuluzi ahlukene e-PCR kuyahluka kakhulu, ngakho-ke kuyanconywa ukuthi kulungiselelwe izimo zokusabela ezilungile zensimbi ethile esheshayo ye-PCR.

2.Uhlelo luvumelana nezimo kakhulu, lunokucaciswa okuphezulu nokuzwela.

3.Ifanele ukusetshenziswa njengama-reagents e-PCR azwela kakhulu, futhi ingasetshenziswa ekuphenduleni kokukhulisa i-PCR nge-multiplex.

4.5′→3′ umsebenzi we-polymerase, 5′→3′ umsebenzi we-exonuclease;akukho 3′→5′ umsebenzi we-exonuclease;awukho umsebenzi wokuhlola amaphutha.

5.Ifanele ukuhlolwa kwekhwalithi nenani le-PCR ne-RT-PCR.

6.Isiphetho esingu-3′ somkhiqizo we-PCR ngu-A, esingahlanganiswa ngokuqondile sibe yi-T vector.

7.Indlela yezinyathelo ezintathu iyanconywa kuma-primer anamazinga okushisa aphansi okushisa noma ukukhulisa izingcezu ezinde kuno-200 bp.