M-MLV Inyandikomvugo
RevScript Reverse transcriptase iboneka hakoreshejwe tekinoroji yubuhanga.Ifite ubushobozi bwa cDNA bwo hejuru, imbaraga zumuriro nubushyuhe bwubushyuhe (kugeza kuri 60 ° C).Igicuruzwa cDNA ikomatanya igera kuri 10 kb.Itezimbere isano yinyandikorugero kandi irakwiriye kwandukura inyandikorugero ya RNA hamwe nuburyo bugoye bwa kabiri cyangwa genes nke za kopi.
Ibigize
| Ibigize | HC2003B-01 (10,000U) | HC2003B-02 (5 * 10,000U) | HC2003B-03 (200.000U) |
| RevScript Guhindura Inyandiko (200U / μL) | 50 μL | 5 × 50 μL | 1 mL |
| 5 × RevScript Buffer | 250 μL | 1.25 mL | 5 mL |
Imiterere y'Ububiko
Ibicuruzwa bigomba kubikwa kuri -25 ° C ~ -15 ° C kumyaka 2.
Igisobanuro
Igice kimwe cyinjizamo nmol 1 ya dTTP mubintu bitangirika aside muminota 10 kuri 37 ° C ukoresheje Oligo (dT) nka primers.
Gushiraho
1.Kwerekana inyandikorugero ya RNA (Iyi ntambwe nubushake, gutandukanya inyandikorugero ya RNA ifasha gufungura ibyiciro bya kabiri, bizamura umusaruro wumurongo wambere cDNA.)
| Ibigize | Umubumbe (μL) |
| RNase kubuntu ddH2O | Kuri 13 |
| Oligo (dT)18 (50 μ mol / L) cyangwa Primer Primer (50 μmol / L) Cyangwa Gene yihariye yibanze (2 μmol / L) | 1 |
| cyangwa 1 | |
| cyangwa 1 | |
| Inyandikorugero ya RNA | X.a |
Inyandiko:
1) a: RNA yose: 1-5 ug cyangwa mRNA: 1-500 ng
2) Kwiyongera kuri 65 ° C muminota 5, hanyuma kwimura kurubura ako kanya kugirango ukonje muminota 2.Muri make centrifugation yo gukusanya reaction ya reaction, ongeramo reaction ya reaction reaction nkuko bigaragara mumbonerahamwe ikurikira.Witonze witonze kuvanga.
1.Gutegura kuvanga reaction (ingano ya 20 μL)
| Ibigize | Umubumbe (μL) |
| Uruvange rwintambwe ibanza | 13 |
| 5 × Buffer | 4 |
| DNTP ivanga (10nmol / L) | 1 |
| Guhindura Inyandiko (200 U / μL) | 1 |
| Inhibitor ya RNase (40 U / μL) | 1 |
1.Kora ibisubizo mubihe bikurikira:
| Ubushyuhe (° C) | Igihe |
| 25 ° C.a | 5min |
| 42 ° C.b | 15-30min |
| 85 ° C.c | 5min |
Inyandiko:
1) a.Kwiyongera kuri 25 ° C kuri 5mins birasabwa gusa kugirango ukoreshe hexamers idasanzwe.Nyamuneka simbuka iyi ntambwe mugihe ukoresheje Oligo (dT)18cyangwa Gene yihariye.
2) b.Ubushuhe busabwa bwo kwandukura ubushyuhe ni 42 ° C, Kubishusho bifite imiterere igoye ya kabiri cyangwa ibintu byinshi bya GC, birasabwa kuzamura ubushyuhe bwa reaction kuri 50-55 ° C.
3) c.Gushyushya kuri 85 ° C kuri 5mins kugirango udakora transcriptase.
4) Ibicuruzwa birashobora gukoreshwa muburyo butaziguye muri PCR cyangwa qPCR, cyangwa bikabikwa kuri -20 ° C kubikwa igihe gito.Birasabwa guhuza ibicuruzwa no kubika kuri -80 ° C kugirango ubike igihe kirekire.Irinde gukonjesha kenshi.
5) Igicuruzwa gikwiranye nintambwe imwe RT-qPCR, birasabwa kongeramo 10-20 U reverse transcriptase kuri buri sisitemu ya reaction ya 25μL, cyangwa kongera buhoro buhoro umubare winyandiko-mvugo ukurikije uko ibintu bimeze.
Inyandiko
1.Nyamuneka saba ahantu ho kugerageza;Gants nziza hamwe na masike bigomba kwambara mugihe cyo gukora.Ibikoreshwa byose byakoreshejwe mubigeragezo bigomba kuba RNase kubuntu kugirango wirinde kwanduza RNase.
2.Uburyo bwose bugomba gukorerwa kurubura kugirango birinde kwangirika kwa RNA.
3.Icyitegererezo cyiza cya RNA kirasabwa kwemeza imikorere ihanitse yo kwandukura.
4.Ibicuruzwa nibikoreshwa mubushakashatsi gusa.
5. Nyamuneka kora ikoti ya laboratoire hamwe na gants imwe ikoreshwa, kubwumutekano wawe.
