M-MLV Reverse Transcriptase
I-RevScript Reverse transcriptase ifunyanwa ngeteknoloji yobunjineli bemfuza.Inamandla aphezulu e-cDNA synthesis, ukuzinza kwe-thermal kunye nomda wokushisa wokuphendula (ukuya kuma-60 ° C).Imveliso ye-cDNA ehlanganisiweyo ifikelela kwi-10 kb.Iphucula ukuhambelana kweetemplates kwaye ifanelekile ukukhutshelwa umva kweetemplate ze-RNA kunye nesakhiwo sesibini esiyinkimbinkimbi okanye ikopi ephantsi yofuzo.
Amacandelo
| Icandelo | HC2003B (10,000U) | HC2003B (5*10,000U) | HC2003B (200,000U) |
| IRevScript Reverse Transcriptase (200U/μL) | 50 μL | 5×50 μL | 1 ml |
| 5 × RevScript Buffer | 250 μL | 1.25 mL | 5 ml |
Imeko yoGcino
Le mveliso kufuneka igcinwe kwi -25°C~-15°C iminyaka emi-2.
Inkcazelo yeyunithi
Iyunithi enye idibanisa i-1 nmol ye-dTTP kwizinto ezingenayo i-asidi kwimizuzu eyi-10 kwi-37 ° C isebenzisa i-Oligo (dT) njenge-primers.
Ukuseta iRection
1.I-Denaturation ye-template ye-RNA (Eli nyathelo liyi-optional, i-denaturation ye-template ye-RNA inceda ukuvula izakhiwo zesibini, eziza kuphucula isivuno se-cDNA yokuqala yomtya.)
| Amacandelo | Umthamo (μL) |
| RNase free ddH2O | Ukuya kwi-13 |
| I-Oligo(dT)18 (50 μmol/L) okanye iPrimer Random (50 μmol/L) Okanye iGene Specific Primes (2 μmol/L) | 1 |
| okanye 1 | |
| okanye 1 | |
| Ithempleyithi ye-RNA | X a |
Amanqaku:
1) a: Iyonke i-RNA: 1-5 ug okanye mRNA: 1-500 ng
2) Ukufukamela kwi-65 ° C imizuzu emi-5, emva koko udlulisele kwiqhwa ngokukhawuleza ukuba ubanda imizuzu emi-2.I-centrifugation emfutshane ukuqokelela ulwelo lokusabela, yongeza isisombululo sempendulo yombhalo ongemva njengoko kubonisiwe kwitheyibhile elandelayo.Ngobumnene i-pipette ukuxuba.
1.Ukulungiswa komxube wokusabela (umthamo we-20 μL)
| Amacandelo | Umthamo (μL) |
| Umxube wenyathelo langaphambili | 13 |
| 5×Isithinteli | 4 |
| Umxube we-dNTP (10nmol/L) | 1 |
| UReverse Transcriptase (200 U/μL) | 1 |
| I-RNase inhibitor (40 U/μL) | 1 |
1.Yenza impendulo phantsi kwezi meko zilandelayo:
| Ubushushu (°C) | Ixesha |
| 25 °Ca | 5 imiz |
| 42 °Cb | 15-30mins |
| 85 °Cc | 5 imiz |
Amanqaku:
1) a.Ukufukamela kwi-25°C kwi-5mins ifuneka kuphela xa usebenzisa i-random hexamers.Nceda utsibe eli nyathelo xa usebenzisa i-Oligo (dT)18okanye iGene Specific Primer.
2) b.Iqondo lokushisa elicetyiswayo lokukhuphela umva liyi-42 ° C, kwiitemplates ezinezakhiwo ezintsonkothileyo zesibini okanye umxholo ophezulu weGC, kuyacetyiswa ukuba unyuse ubushushu bokusabela kwi-50-55 ° C.
3) c.Ukufudumeza kwi-85 ° C kwi-5mins ukuze kusebenze i-reverse transcriptase.
I-4) Imveliso ingasetyenziswa ngokuthe ngqo kwi-PCR okanye kwi-qPCR reactions, okanye igcinwe kwi--20 ° C yokugcina ixesha elifutshane.Kucetyiswa ukuba i-aliguot imveliso kwaye igcine kwi -80 ° C yokugcina ixesha elide.Kuphephe ukunyibilika rhoqo komkhenkce.
5) Imveliso ifanelekile kwinyathelo elinye le-RT-qPCR, kucetyiswa ukuba ungeze i-10-20 U reverse transcriptase kuyo yonke inkqubo yokusabela ye-25μL, okanye ukwandisa ngokuthe ngcembe inani le-reverse transcriptase ngokwemeko yangempela.
Amanqaku
1.Nceda ugcine indawo yovavanyo icocekile;Iiglavu ezicocekileyo kunye nemaski kufuneka zinxitywe ngexesha lokusebenza.Zonke izinto ezisetyenziswayo ezisetyenzisiweyo kuvavanyo kufuneka zikhululeke i-RNase ukunqanda ungcoliseko lwe-RNase.
2.Zonke iinkqubo kufuneka zenziwe kumkhenkce ukuthintela ukuthotywa kweRNA.
3.Iisampulu ezikumgangatho ophezulu we-RNA ziyacetyiswa ukuqinisekisa ukusebenza kakuhle kokukhutshelwa umva.
4.Le mveliso isetyenziselwa uphando kuphela.
5.Nceda usebenzise iidyasi zelebhu kunye neeglavu ezilahlwayo, ukuze ukhuseleke.
