M-MLV Neoscript Ihinduranya Inyandiko
Neoscript Reverse Transcriptase ni transcript transcript yabonetse mugusuzuma mutation ya gene M-MLV ya virusi ya Moloney murine leukemia ikomoka no kugaragara muri E.coli.Enzyme ikuraho ibikorwa bya RNase H, ifite kwihanganira ubushyuhe bwinshi, kandi ikwiranye nubushyuhe bwo hejuru bwo kwandukura.Kubwibyo, ni byiza gukuraho ingaruka mbi ziterwa na RNA yo murwego rwohejuru hamwe nibintu bidasanzwe kuri synthesis ya cDNA, kandi ifite ituze ryinshi hamwe nubushobozi bwo guhinduranya inyandiko.Enzyme ifite ituze ryinshi hamwe nubushobozi bwo guhinduranya inyandiko.
Ibigize
1.200 U / μL Neoscript Ihinduranya Inyandiko
2.5 × Buffer Yambere-Yambere (itabishaka)
* 5 × Buffer Yambere-Yambere ntabwo irimo DNTP, nyamuneka ongeraho DNTP mugihe utegura sisitemu yo kwitwara
Gusabwa
1.Intambwe imwe qRT-PCR.
2.Kumenya virusi ya RNA.
Imiterere y'Ububiko
-20 ° C kubikwa igihe kirekire, bigomba kuvangwa neza mbere yo kubikoresha, irinde gukonjesha kenshi.
Igisobanuro
Igice kimwe kirimo nmol 1 ya dTTP muminota 10 kuri 37 ° C ukoresheje poly (A) • oligo (dT)25nk'icyitegererezo / primer.
Kugenzura ubuziranenge
1.SDS-PAGE isuku ya electrophoreque irenga 98%.
2.Amplification sensitivite, icyiciro-kuri-kugenzura, gutuza.
3.Ntabwo ibikorwa bya nuclease bidasanzwe, nta endogenuclease ya exogenous cyangwa exonuclease yanduye
Igenamigambi Ryakorewe Urunigi Rwa mbere Igisubizo
1.Gutegura kuvanga reaction
| Ibigize | Umubumbe |
| Oligo (dT)12-18 Primer cyangwa Ibisanzwea Cyangwa Gene yihariyeb | 50 pmol |
| 50 pmol (20-100 pmol) | |
| Saa mbiri za mugitondo | |
| 10 mM DNTP | 1 μL |
| Inyandikorugero RNA | RNA≤ 5μg;mRNA≤ 1 μg |
| RNase idafite dH2O | Kuri 10 μL |
Inyandiko:a / b: Nyamuneka hitamo ubwoko butandukanye bwa primers ukurikije ibyo ukeneye kugerageza.
2.Shyushya kuri 65 ° C kuri 5mins hanyuma ukonje vuba kurubura kuri 2mins.
3.Ongeramo ibice bikurikira muri sisitemu yavuzwe haruguru mubunini bwa 20µL hanyuma uvange witonze:
| Ibigize | Umubumbe (μL) |
| 5 Buffer Yambere | 4 |
| Neoscript Guhindura Inyandiko (200 U / μL) | 1 |
| Inhibitor ya RNase (40 U / μL) | 1 |
| RNase idafite dH2O | Kuri 20 μL |
4. Nyamuneka kora reaction ukurikije ibihe bikurikira:
(1) Niba Primer Primer ikoreshwa, reaction igomba gukorwa kuri 25 ℃ kuri 10mins, hanyuma kuri 50 ℃ kuri 30 ~ 60mins;
(2) Niba hakoreshejwe Oligo dT cyangwa primers yihariye, reaction igomba gukorwa kuri 50 ℃ kuri 30 ~ 60mins.
5.Shyushya kuri 95 ℃ kuri 5mins kugirango udakora Neoscript Reverse Transcriptase no guhagarika reaction.
6.Ibicuruzwa byandukuwe birashobora gukoreshwa muburyo bwa PCR hamwe na fluorescence igereranya PCR reaction, cyangwa ikabikwa kuri -20 ℃ igihe kirekire.
PCR R.reaction ：
1.Gutegura kuvanga reaction
| Ibigize | Kwibanda |
| 10 × Buffer ya PCR (DNTP kubuntu, Mg² + ubuntu) | 1 × |
| DNTPs (10mM buri dNTP) | 200 μM |
| 25 mM MgCl2 | 1-4 mM |
| Taq ADN Polymerase (5U / μL) | 2-2.5 U. |
| Primer 1 (10 μM) | 0.2-1 μM |
| Primer 2 (10 μM) | 0.2-1 μM |
| Inyandikorugeroa | ≤10% Igisubizo cya mbere cyurunigi (2 μL) |
| ddH2O | Kuri 50 μL |
Inyandiko:a: Niba haribintu byinshi byambere byakemuwe byongeweho, PCR irashobora kubuzwa.
2.Uburyo bwa PCR
| Intambwe | Ubushyuhe | Igihe | Amagare |
| Mbere yo gutandukana | 95 ℃ | Iminota 2-5 | 1 |
| Gutandukana | 95 ℃ | Amasegonda 10-20 | 30-40 |
| Annealing | 50-60 ℃ | Amasegonda 10-30 | |
| Kwagura | 72 ℃ | Amasegonda 10-60 |
Inyandiko
1.Birakwiriye guhinduranya ubushyuhe bwo guhinduranya ubushyuhe buri hagati ya 42 ℃ ~ 55 ℃.
2.Ifite ituze ryiza, ikwiranye nubushyuhe bwo hejuru reaction transcription amplification.Byongeye kandi, nibyiza kunyura neza mubice bigoye byubaka bya RNA.Kandiikwiranye nintambwe imwe multiplex fluorescence igereranya RT-PCR.
3.Guhuza neza na enzymes zitandukanye za PCR kandi birakwiriye kubyumva cyane RT-PCR.
4.Birakwiye kubyunvikana cyane intambwe imwe ya fluorescence igereranya RT-PCR reaction, bizamura neza igipimo cyo gutahura kwinshi kwicyitegererezo.
5.Birakwiriye kubaka isomero rya cDNA.
