M-MLV Reverse Transcriptase
RevScript Reverse transcriptase ana samun su ta fasahar injiniyan kwayoyin halitta.Yana da ƙarfin haɗin cDNA mafi girma, kwanciyar hankali na thermal da iyakar zafin jiki (har zuwa 60 ° C).Samfurin cDNA da aka haɗa ya kai 10kb.Yana haɓaka alaƙar samfuran kuma ya dace da jujjuya rubutun samfuran RNA tare da hadadden tsari na sakandare ko ƙananan kwafi.
Abubuwan da aka gyara
| Bangaren | HC2003B (10,000U) | HC2003B (5*10,000U) | HC2003B (200,000U) |
| RevScript Reverse Transcriptase (200U/μL) | 50 μl | 5 × 50 ml | 1 ml |
| 5 × RevScript Buffer | 250 ml | 1.25 ml | ml 5 |
Yanayin Ajiya
Ya kamata a adana wannan samfurin a -25 ° C ~ -15 ° C na shekaru 2.
Ma'anar Naúrar
Raka'a ɗaya ta haɗa 1 nmol na dTTP cikin kayan acid-insoluble a cikin mintuna 10 a 37°C ta amfani da Oligo(dT) a matsayin masu farawa.
Saitin Amsa
1.Denaturation na RNA samfuri (Wannan mataki na zaɓi ne, ƙirƙira samfurin RNA yana taimakawa buɗe tsarin na biyu, wanda zai haɓaka yawan amfanin farko na cDNA.)
| Abubuwan da aka gyara | girma (μL) |
| RNase kyauta ddH2O | Zuwa 13 |
| Oligo(dT)18 (50 μmol/L) ko Random Primer (50 μmol/L) Ko Takaddun Matsalolin Halitta (2 μmol/L) | 1 |
| ko 1 | |
| ko 1 | |
| Samfurin RNA | X a |
Bayanan kula:
1) a: Jimlar RNA: 1-5 ug ko mRNA: 1-500 ng
2) Sanya a 65 ° C na minti 5, sa'an nan kuma canjawa kan kankara nan da nan don yin sanyi na minti 2.Taƙaitaccen centrifugation don tattara ruwa mai amsawa, ƙara bayanin amsawar juyi kamar yadda aka nuna a tebur mai zuwa.A hankali pipette don haɗuwa.
1.Shirye-shiryen cakuda amsa (ƙarar 20 μL)
| Abubuwan da aka gyara | girma (μL) |
| Cakuda matakin da ya gabata | 13 |
| 5× Buffer | 4 |
| dNTP Mix (10nmol/L) | 1 |
| Rubutun Juya (200 U/μL) | 1 |
| Mai hana RNase (40 U/μL) | 1 |
1.Yi martani a ƙarƙashin sharuɗɗa masu zuwa:
| Zazzabi (°C) | Lokaci |
| 25 °Ca | 5 min |
| 42 °Cb | 15-30 min |
| 85 °Cc | 5 min |
Bayanan kula:
1) a.Ana buƙatar haɓakawa a 25°C na 5mins kawai don amfani da hexamers bazuwar.Da fatan za a tsallake wannan matakin lokacin amfani da Oligo (dT)18ko Gene Specific Primer.
2) b.Matsakaicin zafin juzu'i da aka ba da shawarar shine 42°C, Don samfura masu sarƙaƙƙiya tsarin sakandare ko babban abun ciki na GC, ana ba da shawarar ɗaga yawan zafin jiki zuwa 50-55°C.
3) c.Dumama a 85°C na tsawon mintuna 5 don hana juyar da rubutu.
4) Ana iya amfani da samfurin kai tsaye a cikin halayen PCR ko qPCR, ko adana shi a -20°C don ajiyar ɗan gajeren lokaci.An ba da shawarar a daidaita samfuran kuma adana a -80 ° C don adana dogon lokaci.Guji daskarewa akai-akai.
5) Samfurin ya dace da mataki ɗaya na RT-qPCR, ana bada shawara don ƙara 10-20 U reverse transcriptase ga kowane tsarin amsawa na 25μL, ko a hankali ƙara adadin juzu'in juzu'i bisa ga ainihin halin da ake ciki.
Bayanan kula
1.Da fatan za a tsabtace wurin gwaji;Ya kamata a sanya safofin hannu masu tsabta da abin rufe fuska yayin aiki.Duk abubuwan da ake amfani da su a cikin gwajin yakamata su kasance masu 'yanci na RNase don hana gurɓacewar RNase.
2.Dole ne a yi dukkan hanyoyin akan kankara don hana lalata RNA.
3.Ana ba da shawarar samfuran RNA masu inganci don tabbatar da ingantaccen juzu'i.
4.Wannan samfurin don amfanin bincike ne kawai.
5.Da fatan za a yi aiki da riguna na lab da safofin hannu masu yuwuwa, don amincin ku.
