M-MLV Neoscript Reverse Transcriptase
Neoscript Reverse Transcriptase ndi reverse transcriptase yopezedwa mwa mutation screening ya M-MLV jini ya Moloney murine leukemia virus chiyambi ndi mawu mu E.coli.Enzyme imachotsa ntchito ya RNase H, imakhala yolekerera kutentha kwambiri, ndipo ndiyoyenera kulembedwa mosintha kutentha kwambiri.Choncho, ndizothandiza kuthetsa zotsatira zoipa za mawonekedwe apamwamba a RNA ndi zinthu zomwe sizinali zenizeni pa kaphatikizidwe ka cDNA, ndipo zimakhala ndi kukhazikika kwapamwamba komanso kusinthika kwa kaphatikizidwe ka mawu.Enzyme ili ndi kukhazikika kwapamwamba komanso kuthekera kosinthira kumasulira.
Zigawo
1.200 U/μL Neoscript Reverse Transcriptase
2.5 × First-Strand Buffer (posankha)
* 5 × First-Strand Buffer ilibe dNTP, chonde onjezani ma dNTP pokonzekera machitidwe
Ntchito yovomerezeka
1.Gawo limodzi qRT-PCR.
2.Kuzindikira kachilombo ka RNA.
Mkhalidwe Wosungira
-20 ° C posungira nthawi yayitali, ziyenera kusakanizidwa bwino musanagwiritse ntchito, pewani kuzizira pafupipafupi.
Tanthauzo la Chigawo
Chigawo chimodzi chimaphatikiza 1 nmol ya dTTP mu mphindi 10 pa 37°C pogwiritsa ntchito poly(A)•oligo(dT)25monga template / primer.
Kuwongolera Kwabwino
1.SDS-PAGE electrophoretic chiyero choposa 98%.
2.Kukhudzika kukhudzika, kuwongolera kwa batch-to-batch, kukhazikika.
3.Palibe exogenous nuclease ntchito, palibe exogenous endonuclease kapena exonuclease kuipitsidwa
Kukonzekera kwa Reaction kwa First Chain Reaction Solution
1.Kukonzekera anachita osakaniza
| Zigawo | Voliyumu |
| Oligo (dT)12-18 Choyamba kapena Random Primera Kapena Gene Specific Primersb | 50 pml |
| 50 pmol (20-100 pmol) | |
| 2 pmw | |
| 10 mm dNTP | 1 ml |
| Chithunzi cha RNA | Zonse za RNA≤ 5μg;mRNA≤ 1 μg |
| RNase-free dH2O | mpaka 10 μl |
Ndemanga:a/b: Chonde sankhani mitundu yoyambira yamitundu yosiyanasiyana malinga ndi zosowa zanu zoyeserera.
2.Kutenthetsa pa 65 ° C kwa 5mins ndikuzizira mofulumira pa ayezi kwa 2mins.
3.Onjezani magawo otsatirawa pamakina omwe ali pamwambapa ku voliyumu yonse ya 20µL ndikusakaniza mofatsa:
| Zigawo | Voliyumu (μL) |
| 5 × Choyamba-Strand Buffer | 4 |
| Neoscript Reverse Transcriptase (200 U/μL) | 1 |
| RNase inhibitor (40 U/μL) | 1 |
| RNase-free dH2O | mpaka 20 μl |
4.Chonde chitani zomwezo motengera izi:
(1) Ngati Random Primer ikugwiritsidwa ntchito, zomwe ziyenera kuchitika pa 25 ℃ kwa 10mins, ndiyeno pa 50 ℃ kwa 30 ~ 60mins;
(2) Ngati Oligo dT kapena zoyambira enieni ntchito, anachita ayenera kuchitidwa pa 50 ℃ kwa 30 ~ 60mins.
5.Kutenthetsa pa 95 ℃ kwa 5mins kuti mutsegule Neoscript Reverse Transcriptase ndikuthetsa zomwe zikuchitika.
6.Zolemba zosinthira zitha kugwiritsidwa ntchito mwachindunji mu PCR reaction ndi fluorescence quantitative PCR reaction, kapena kusungidwa pa -20 ℃ kwa nthawi yayitali.
PCR Rkuchita:
1.Kukonzekera anachita osakaniza
| Zigawo | Kukhazikika |
| 10 × PCR Buffer (dNTP yaulere, Mg²+ yaulere) | 1 × pa |
| dNTPs (10mM iliyonse dNTP) | 200 μM |
| 25 mm MgCl2 | 1-4 mm |
| Taq DNA Polymerase (5U/μL) | 2-2.5 U |
| Woyamba 1 (10 μM) | 0.2-1 μM |
| Woyamba 2 (10 μM) | 0.2-1 μM |
| Templatea | ≤10% First Chain Reaction Solution (2 μL) |
| ddH2O | mpaka 50 μl |
Ndemanga:a: Ngati njira yoyamba yowonjezera yowonjezera iwonjezeredwa, machitidwe a PCR akhoza kuletsedwa.
2.PCR Reaction Procedure
| Khwerero | Kutentha | Nthawi | Zozungulira |
| Pre-denaturation | 95 ℃ | 2-5 min | 1 |
| Denaturation | 95 ℃ | 10-20 sec | 30-40 |
| Annealing | 50-60 ℃ | 10-30 sec | |
| Kuwonjezera | 72 ℃ | 10-60 sec |
Zolemba
1.Zoyenera kukhathamiritsa kutentha kwa 42 ℃ ~ 55 ℃.
2.Ili ndi kukhazikika bwino, ndiyoyenera kukulitsa kutentha kwa reverse transcript.Kuphatikiza apo, ndi yabwino kudutsa bwino m'magawo ovuta a RNA.Komanso, izondiyoyenera kuzindikirika kwa gawo limodzi la multiplex fluorescence kuchuluka kwa RT-PCR.
3.Kugwirizana kwabwino ndi michere yokulitsa ya PCR ndipo ndikoyenera kuchitapo kanthu kwa RT-PCR.
4.Oyenera kukhudzika kwambiri sitepe imodzi ya fluorescence kuchuluka kwa RT-PCR reaction, imathandizira bwino kuzindikirika kwa ma tempuleti otsika.
5.Zoyenera kupanga laibulale ya cDNA.
