M-MLV Reverse Transcriptase
RevScript Reverse transcriptase e fumanoa ka theknoloji ea liphatsa tsa lefutso.E na le bokhoni bo phahameng ba ho kopanya cDNA, botsitso ba mocheso le moeli oa mocheso oa karabelo (ho fihla ho 60 ° C).Sehlahisoa sa maiketsetso sa cDNA se fihla ho 10 kb.E matlafatsa kamano ea lithempleite 'me e loketse ho ngoloa ka morao ha litempele tsa RNA tse nang le sebopeho se rarahaneng sa bobeli kapa liphatsa tsa lefutso tse tlase.
Likaroloana
| Karolo | 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 |
Boemo ba polokelo
Sehlahisoa sena se lokela ho bolokoa ho -25°C~-15°C ka lilemo tse 2.
Tlhaloso ea Yuniti
Yuniti e le 'ngoe e kenyelletsa 1 nmol ea dTTP linthong tse sa keneng acid ka metsotso e 10 ho 37 ° C ho sebelisa Oligo(dT) joalo ka li-primers.
Rection Setup
1.Denaturation ea template ea RNA (Mohato ona ke oa boikhethelo, denaturation ea template ea RNA e thusa ho bula mehaho ea bobeli, e tla ntlafatsa chai ea cDNA ea pele.)
| Likaroloana | Bolumo (μL) |
| RNase mahala ddH2O | Ho ea ho 13 |
| Oligo(dT)18 (50 μmol/L) kapa Primer e sa reroang (50 μmol/L) Kapa Gene Specific Primers (2 μmol/L) | 1 |
| kapa 1 | |
| kapa 1 | |
| Setšoantšo sa RNA | X a |
Lintlha:
1) a: Kakaretso ea RNA: 1-5 ug kapa mRNA: 1-500 ng
2) Ho kenya ka 65 ° C metsotso e 5, ebe o fetisetsa leqhoa hang-hang ho bata metsotso e 2.Bokhutšoane ba centrifugation ho bokella metsi a reaction, eketsa tharollo ea reverse transcript joalo ka ha ho bonts'itsoe lethathamong le latelang.Ka bonolo pipette ho kopanya.
1.Ho lokisa motsoako oa karabelo (20 μL bophahamo ba modumo)
| Likaroloana | Bolumo (μL) |
| Motsoako oa mohato o fetileng | 13 |
| 5× Buffer | 4 |
| dNTP Mix (10nmol/L) | 1 |
| Reverse Transcriptase (200 U/μL) | 1 |
| RNase inhibitor (40 U/μL) | 1 |
1.Etsa karabelo tlas'a maemo a latelang:
| Mocheso (°C) | Nako |
| 25 °Ca | 5 mets |
| 42 °Cb | 15-30mins |
| 85 °Cc | 5 mets |
Lintlha:
1) a.Ho kenya 25°C ka metsotso e 5 ho hlokahala feela bakeng sa ho sebelisa li-hexamers tse sa tloaelehang.Ka kopo, tlola mohato ona ha u sebelisa Oligo (dT)18kapa Gene Specific Primer.
2) b.Mocheso o khothaletsoang ka morao ke 42 ° C, Bakeng sa litempele tse nang le meaho e rarahaneng ea bobeli kapa litaba tse phahameng tsa GC, ho khothaletsoa ho phahamisa mocheso oa karabelo ho 50-55 ° C.
3) c.E futhumala ho 85°C bakeng sa metsotso e 5 ho etsa hore reverse transcriptase e sebetse.
4) Sehlahisoa se ka sebelisoa ka kotloloho ho liketso tsa PCR kapa qPCR, kapa tsa bolokoa ho -20 ° C bakeng sa polokelo ea nakoana.Ho khothalletsoa ho kopanya lihlahisoa le ho boloka ho -80 ° C bakeng sa polokelo ea nako e telele.Qoba ho qhibiliha khafetsa.
5) Sehlahisoa se loketse mohato o le mong oa RT-qPCR, ho kgothaletswa ho eketsa 10-20 U reverse transcriptase bakeng sa tsamaiso e 'ngoe le e' ngoe ea 25μL, kapa butle-butle eketsa palo ea reverse transcriptase ho ea ka boemo ba sebele.
Lintlha
1.Ka kopo boloka sebaka sa liteko se hloekile;Li-gloves le limaske tse hloekileng li lokela ho apesoa nakong ea ts'ebetso.Lisebelisoa tsohle tse sebelisitsoeng tekong li lokela ho ba mahala tsa RNase ho thibela tšilafalo ea RNase.
2.Mekhoa eohle e lokela ho etsoa holim'a leqhoa ho thibela ho senyeha ha RNA.
3.Ho khothaletsoa lisampole tsa boleng bo holimo tsa RNA ho netefatsa ts'ebetso e phahameng ea ho fetolela ka morao.
4.Sehlahisoa sena ke sa tšebeliso ea lipatlisiso feela.
5.Ka kopo sebetsa ka lijase tsa lab le liatlana tse lahloang, molemong oa polokeho ea hau.
