M-MLV Reverse Transcriptase
RevScript Reverse transcriptase imapezeka ndi ukadaulo wa genetic engineering.Ili ndi luso lapamwamba la kaphatikizidwe ka cDNA, kukhazikika kwamafuta ndi malire a kutentha (mpaka 60 ° C).Zomwe zimapangidwa ndi cDNA zimatha kufika 10 kb.Imakulitsa kuyanjana kwa ma templates ndipo ndiyoyenera kulemberanso ma tempulo a RNA okhala ndi mawonekedwe achiwiri ovuta kapena ma gene otsika.
Zigawo
| Chigawo | HC2003B (10,000U) | HC2003B (5 * 10,000U) | HC2003B (200,000U) |
| RevScript Reverse Transcriptase (200U/μL) | 50 μl pa | 5 × 50 μL | 1 ml |
| 5 × RevScript Buffer | 250 μl | 1.25 mL | 5ml pa |
Mkhalidwe Wosungira
Izi ziyenera kusungidwa pa -25°C~-15°C kwa zaka ziwiri.
Tanthauzo la Chigawo
Chigawo chimodzi chimaphatikizapo 1 nmol ya dTTP muzinthu zosasungunuka za asidi mu mphindi 10 pa 37 ° C pogwiritsa ntchito Oligo(dT) monga zoyambira.
Kukonzekera kwa Reaction
1.Denaturation ya RNA template (Izi ndizosankha, kutanthauzira kwa template ya RNA kumathandizira kutsegula zida zachiwiri, zomwe zingapangitse zokolola za cDNA yoyamba.)
| Zigawo | Voliyumu (μL) |
| RNase yaulere ddH2O | Ku 13 |
| Oligo (dT)18 (50 μmol/L) kapena Primer Yosasinthika (50 μmol/L) Kapena Gene Specific Primers (2 μmol/L) | 1 |
| kapena 1 | |
| kapena 1 | |
| Chithunzi cha RNA | X a |
Ndemanga:
1) a: RNA yonse: 1-5 ug kapena mRNA: 1-500 ng
2) Kufungatira pa 65 ° C kwa mphindi zisanu, kenako ndikusamutsa pa ayezi nthawi yomweyo kuti muzizire kwa mphindi ziwiri.Mwachidule centrifugation kusonkhanitsa reaction madzi, onjezani reverse transcription reaction yankho monga momwe tebulo ili m'munsiyi.Pang'onopang'ono pipette kusakaniza.
1.Kukonzekera kosakanikirana (20 μL voliyumu)
| Zigawo | Voliyumu (μL) |
| Kusakaniza kwa sitepe yapitayi | 13 |
| 5 × Bafa | 4 |
| dNTP Mix (10nmol/L) | 1 |
| Reverse Transcriptase (200 U/μL) | 1 |
| RNase inhibitor (40 U/μL) | 1 |
1.Chitani zomwezo pansi pamikhalidwe iyi:
| Kutentha (°C) | Nthawi |
| 25 °Ca | 5 mins |
| 42 °Cb | 15-30mins |
| 85 °Cc | 5 mins |
Ndemanga:
1) a.Kuyika pa 25°C kwa 5mins kumafunika kokha pogwiritsa ntchito ma hexamers osasintha.Chonde dumphani izi mukamagwiritsa ntchito Oligo (dT)18kapena Gene Specific Primer.
2) b.Kutentha kovomerezeka kwa reverse transcript ndi 42 ° C, Kwa ma tempuleti okhala ndi zovuta zachiwiri kapena GC zambiri, tikulimbikitsidwa kukweza kutentha kwa 50-55 ° C.
3) c.Kutenthetsa pa 85 ° C kwa 5mins kuti mutsegule reverse transcriptase.
4) Mankhwalawa angagwiritsidwe ntchito mwachindunji mu machitidwe a PCR kapena qPCR, kapena kusungidwa pa -20 ° C kuti asungidwe kwakanthawi kochepa.Ndikoyenera kuti aliguot mankhwala ndi kusunga pa -80 ° C kwa nthawi yaitali yosungirako.Pewani kuzizira pafupipafupi.
5) Zogulitsazo ndizoyenera gawo limodzi la RT-qPCR, tikulimbikitsidwa kuwonjezera 10-20 U reverse transcriptase pamtundu uliwonse wa 25μL, kapena kuonjezera pang'onopang'ono kuchuluka kwa reverse transcriptase malinga ndi momwe zilili.
Zolemba
1.Chonde sungani malo oyesera;Magolovesi oyera ndi masks ayenera kuvala panthawi yogwira ntchito.Zonse zomwe zimagwiritsidwa ntchito poyesera ziyenera kukhala zaulere za RNase kuti zipewe kuipitsidwa kwa RNase.
2.Njira zonse ziyenera kuchitidwa pa ayezi kuti apewe kuwonongeka kwa RNA.
3.Zitsanzo za RNA zapamwamba kwambiri zimalimbikitsidwa kuti zitsimikizire kuti zolembera zosintha kwambiri zikuyenda bwino.
4.Izi ndizogwiritsidwa ntchito pofufuza zokha.
5.Chonde gwirani ntchito ndi malaya a labu ndi magolovesi otayika, kuti mutetezeke.
