M-MLV Neoscript Reverse Transcriptase
Neoscript Reverse Transcriptase ke reverse transcriptase e fumanoeng ka ho hlahlobisisa phetoho ea mofuta oa M-MLV oa tlhaho ea vaerase ea Moloney murine leukemia le polelo ho E.coli.Enzyme e tlosa tšebetso ea RNase H, e na le mamello e phahameng ea mocheso, 'me e loketse ho ngoloa ha mocheso o phahameng oa mocheso.Ka hona, hoa thusa bakeng sa ho felisa liphello tse sa thabiseng tsa sebopeho sa boemo bo phahameng ba RNA le lintlha tse sa tobang ka cDNA synthesis, 'me e na le botsitso bo phahameng le bokhoni ba ho fetola transcript synthesis.Enzyme e na le botsitso bo phahameng le bokhoni ba ho fetolela transcript.
Likaroloana
1.200 U/μL Neoscript Reverse Transcriptase
2.5 × First-Strand Buffer (ho ikhethela)
* 5 × First-Strand Buffer ha e na dNTP, ka kopo eketsa dNTPs ha u lokisa mokhoa oa ho arabela.
E khothalelitsoeng Kopo
1.Mohato o le mong qRT-PCR.
2.Ho fumanoa ha vaerase ea RNA.
Boemo ba polokelo
-20°C bakeng sa polokelo ea nako e telele, e lokela ho tsoakoa hantle pele e sebelisoa, qoba ho qhibiliha khafetsa.
Tlhaloso ea Yuniti
Yuniti e le 'ngoe e kenyelletsa 1 nmol ea dTTP ka metsotso e 10 ho 37°C e sebelisa poly(A)•oligo(dT)25joalo ka template/primer.
Taolo ea Boleng
1.SDS-PAGE electrophoretic purity e kholo ho feta 98%.
2.Amplification sensitivity, taolo ea batch-to-batch, botsitso.
3.Ha ho ts'ebetso ea "nuclease exogenous", ha ho na endonuclease ea kantle kapa tšilafalo ea exonuclease.
Rection Setup bakeng sa First Chain Reaction Solution
1.Ho itokisetsa motsoako oa karabelo
| Likaroloana | Bolumo |
| Oligo(dT)12-18 Se qalang kapa Primer e sa reroanga Kapa Gene Specific Primersb | 50 pmol |
| 50 pmol (20-100 pmol) | |
| 2 pmol | |
| 10 mM dNTP | 1 μL |
| Setšoantšo sa RNA | Kakaretso ea RNA≤ 5μg;mRNA≤ 1 μg |
| RNase-free dH2O | Ho isa ho 10 μL |
Lintlha:a/b: Ka kopo khetha mefuta e fapaneng ea li-primer ho latela litlhoko tsa hau tsa liteko.
2.Chesa ka 65 ° C bakeng sa 5mins 'me u pholile ka potlako holim'a leqhoa bakeng sa 2mins.
3.Kenya likarolo tse latelang ho sistimi e kaholimo ho bophahamo ba 20µL 'me u kopanye ka bonolo:
| Likaroloana | Bolumo (μL) |
| 5 × Pele-Strand Buffer | 4 |
| Neoscript Reverse Transcriptase (200 U/μL) | 1 |
| RNase inhibitor (40 U/μL) | 1 |
| RNase-free dH2O | Ho isa ho 20 μL |
4.Ka kopo etsa karabelo ho latela maemo a latelang:
(1) Haeba Random Primer e sebelisoa, karabelo e lokela ho etsoa ho 25 ℃ bakeng sa 10mins, ebe ho 50 ℃ bakeng sa 30 ~ 60mins;
(2) Haeba Oligo dT kapa li-primers tse itseng li sebelisoa, karabelo e lokela ho etsoa ka 50 ℃ bakeng sa 30 ~ 60mins.
5.Chesa ho 95 ℃ bakeng sa 5mins ho tima Neoscript Reverse Transcriptase le ho felisa karabelo.
6.Lihlahisoa tsa reverse transcription li ka sebelisoa ka kotloloho ho PCR reaction le fluorescence quantitative PCR reaction, kapa tsa bolokoa ho -20 ℃ nako e telele.
PCR Rketso:
1.Ho itokisetsa motsoako oa karabelo
| Likaroloana | Ho tsepamisa maikutlo |
| 10 × PCR Buffer (dNTP mahala, Mg²+ mahala) | 1× |
| dNTPs (10mM ka 'ngoe dNTP) | 200 μM |
| 25 limilimithara MgCl2 | 1-4 mmM |
| Taq DNA Polymerase (5U/μL) | 2-2.5 U |
| Primer 1 (10 μM) | 0.2-1 μM |
| Primer 2 (10 μM) | 0.2-1 μM |
| Setšoantšoa | ≤10% First Chain Reaction Solution (2 μL) |
| ddH2O | Ho fihlela ho 50 μL |
Lintlha:a: Haeba tharollo ea pele ea ketane e ngata haholo e eketsoa, karabelo ea PCR e kanna ea thibeloa.
2.Mokhoa oa ho itšoara ka PCR
| Mohato | Mocheso | Nako | Lipotoloho |
| Pre-denaturation | 95℃ | 2-5 mets | 1 |
| Denaturation | 95℃ | 10-20 sec | 30-40 |
| Annealing | 50-60 ℃ | 10-30 sec | |
| Katoloso | 72℃ | 10-60 sec |
Lintlha
1.E loketse ho ntlafatsa mocheso oa transcript ho 42 ℃ ~ 55 ℃.
2.E na le botsitso bo betere, e loketse bakeng sa amplification e phahameng ea mocheso oa reverse.Ntle le moo, e loketse ho feta hantle libakeng tse rarahaneng tsa RNA.Hape, eonae loketse tlhahlobo ea RT-PCR ea mohato o le mong oa multiplex fluorescence quantitative.
3.E lumellana hantle le li-enzyme tse fapaneng tsa amplification tsa PCR 'me e loketse karabelo e phahameng ea RT-PCR.
4.E loketse kutloisiso e phahameng ea mohato o le mong oa fluorescence quantitative RT-PCR, ka katleho e ntlafatsa sekhahla sa ho lemoha ha mahloriso a tlase a litempele.
5.E loketse kaho ea laebrari ea cDNA.
