M-MLV Reverse Transcriptase
RevScript Reverse transcriptase inowanikwa ne genetic engineering tekinoroji.Iyo ine yakakwirira cDNA synthesis kugona, kugadzikana kwekupisa uye kuita tembiricha muganho (kusvika 60 ° C).Iyo yakagadzirwa cDNA chigadzirwa inosvika gumi kb.Iyo inokwidziridza hukama hwematemplate uye inokodzera reverse transcript yeRNA templates ine yakaoma yechipiri chimiro kana yakaderera kopi majini.
Zvikamu
| Chikamu | HC2003B (10,000U) | HC2003B (5*10,000U) | HC2003B (200,000U) |
| RevScript Reverse Transcriptase (200U/μL) | 50 μL | 5 × 50 μL | 1 mL |
| 5 × RevScript Buffer | 250 μL | 1.25 mL | 5 mL |
Storage Condition
Ichi chigadzirwa chinofanira kuchengetwa pa -25°C~-15°C kwemakore maviri.
Chikamu Tsanangudzo
Imwe yuniti inosanganisa 1 nmol yedTTP muasidhi-insoluble zvinhu mumaminetsi gumi pa37 ° C uchishandisa Oligo (dT) sekutanga.
Reaction Setup
1.Denaturation yeRNA template (Iyi nhanho ndeyekusarudza, denaturation yeRNA template inobatsira kuvhura yechipiri zvimiro, izvo zvinovandudza goho rekutanga strand cDNA.)
| Zvikamu | Vhoriyamu (μL) |
| RNase yemahara ddH2O | Ku13 |
| Oligo(dT)18 (50 μmol/L) kana Random Primer (50 μmol/L) Kana Gene Specific Primes (2 μmol/L) | 1 |
| kana 1 | |
| kana 1 | |
| RNA template | X a |
Notes:
1) a: Yese RNA: 1-5 ug kana mRNA: 1-500 ng
2) Kufurira pa65°C kwemaminitsi mashanu, wozoisa paaizi nekukasira kuti itonhore kwemaminetsi maviri.Muchidimbu centrifugation kuunganidza reaction fluid, wedzera reverse transcription reaction solution sezvinoratidzwa patafura inotevera.Zvinyoro pipette kusanganisa.
1.Kugadzirira kweiyo reaction musanganiswa (20 μL vhoriyamu)
| Zvikamu | Vhoriyamu (μL) |
| Musanganiswa wenhanho yapfuura | 13 |
| 5×Buffer | 4 |
| dNTP Musanganiswa (10nmol/L) | 1 |
| Reverse Transcriptase (200 U/μL) | 1 |
| RNase inhibitor (40 U/μL) | 1 |
1.Ita reaction pasi pemamiriro anotevera:
| Tembiricha (°C) | Nguva |
| 25 °Ca | 5 mins |
| 42 °Cb | 15-30mins |
| 85 °Cc | 5 mins |
Notes:
1) a.Kufukura pa25°C kwe5mins kunodiwa chete pakushandisa ma random hexamers.Ndokumbira svetuka danho iri kana uchishandisa Oligo (dT)18kana Gene Specific Primer.
2) b.Iyo inokurudzirwa reverse transcript tembiricha ndeye 42 ° C, Kune matemplate ane akaomarara echipiri zvimiro kana yakakwira GC zvemukati, zvinokurudzirwa kukwidza kuita tembiricha kusvika 50-55 ° C.
3) c.Kupisa pa85°C kwemaminetsi mashanu kuti isaite reverse transcriptase.
4) Chigadzirwa chacho chinogona kushandiswa zvakananga muPCR kana qPCR maitiro, kana kuchengetwa pa -20 ° C kuitira kuchengetedza kwenguva pfupi.Inokurudzirwa kubatanidza zvigadzirwa uye kuchengeta pa -80 ° C kuitira kuchengetedza kwenguva refu.Dzivisa kugaroomesa nechando.
5) Chigadzirwa chakakodzera kune imwe-nhanho RT-qPCR, inokurudzirwa kuwedzera 10-20 U reverse transcriptase kune yega yega 25μL reaction system, kana zvishoma nezvishoma kuwedzera huwandu hwereverse transcriptase zvinoenderana nemamiriro chaiwo.
Notes
1.Ndapota chengetedza nzvimbo yekuedza yakachena;Magurovhosi akachena uye masikisi anofanirwa kupfekwa panguva yekushanda.Zvese zvinoshandiswa mukuyedza zvinofanirwa kunge zvisina RNase kudzivirira kusvibiswa kweRNase.
2.Maitiro ese anofanirwa kuitwa pachando kudzivirira kuparara kweRNA.
3.Yemhando yepamusoro RNA samples inokurudzirwa kuti ive nechokwadi chepamusoro-soro yekureverse transcription.
4.Ichi chigadzirwa ndechekutsvaga kushandiswa chete.
5.Ndokumbira ushande nemajasi erabhoritari uye magurovhosi anoraswa, kuitira kuchengetedza kwako.
