RTL Reverse Transcriptase
RTL reverse transcriptase shine DNA polymerase mai dogaro da samfuri na RNA wanda ya rasa aikin 3'→5' exonuclease kuma yana da aikin RNase H.Wannan enzyme na iya amfani da RNA a matsayin samfuri don haɗa wani madaidaicin madauri na DNA, wanda za'a iya amfani da shi zuwa haɗin cDNA na farko, musamman don RT-LAMP (madaidaicin isothermal amplification).Idan aka kwatanta da RTL reverse transcriptase 1.0, an inganta hankali sosai, kwanciyar hankali na thermal yana da ƙarfi, kuma amsawa a 65 ° C ya fi kwanciyar hankali.Ana iya amfani da RTL reverse transcriptase (free glycerol) don shirya shirye-shiryen lyophilized, lyophilized RT-LAMP reagents da dai sauransu.
Ma'anar Naúrar
Raka'a ɗaya ta haɗa 1 nmol na dTTP cikin kayan acid-acid a cikin mintuna 20 a 50°C ta amfani da poly(A)•oligo(dT)25 azaman samfuri-primer.
Abubuwan da aka gyara
| Bangaren | HC5008A-01 | HC5008A-02 | HC5008A-03 |
| RTL Reverse Transcriptase (Babu Glycerol) (15U/μL) | 0.1 ml | 1 ml | ml 10 |
| 10 × HC RTL Buffer | 1.5 ml | 4 × 1.5 ml | 5 × 10 ml |
| MgSO4 (100mM) | 1.5 ml | 2 × 1.5 ml | 3 × 10 ml |
Yanayin Ajiya
Jirgin da ke ƙarƙashin 0 ° C kuma a adana shi a -25 ° C ~ -15 ° C.
Kula da inganci
- Ragowar AyyukanEndonuclease:A 50 μL dauki dauke da 1 μg na λDNA da 15 raka'a na RTL2.0 incubated for 16 hours a 37 ℃ nuna iri iri kamar yadda korau iko ta gel electrophoresis.
- Ragowar AyyukanExonuclease:A 50 μL dauki dauke da 1 μg na Hind Ⅲ narkewa λDNA da 15 raka'a na RTL2.0 incubated na 16 hours a 37 ℃ yana nuna iri ɗaya kamar iko mara kyau ta gel electrophoresis.
- Ragowar AyyukanNickase:Halin 50 μL mai ɗauke da 1 μg na supercoiled pBR322 da raka'a 15 na RTL2.0 da aka haɗa na tsawon awanni 4 a 37 ° C yana nuna tsari iri ɗaya azaman iko mara kyau ta gel electrophoresis.
- Ragowar AyyukanRNase:Halin 10 μL mai ɗauke da 0.48 μg na MS2 RNA da raka'a 15 na RTL2.0 da aka haɗa na tsawon awanni 4 a 37°C yana nuna tsari iri ɗaya azaman iko mara kyau ta gel electrophoresis.
- E. coli gDNA:An auna daE.colitakamaiman kayan gano HCD, raka'a 15 na RTL2.0 sun ƙunshi ƙasa da 1E. colikwayoyin halitta.
Saitin Amsa
cDNA Synthesis Protocol
| Abubuwan da aka gyara | Ƙarar |
| Samfura RNA a | na zaɓi |
| Oligo(dT) 18 ~ 25(50uM) ko Random Primer mix(60uM) | 2 μl |
| dNTP Mix (10mM kowane) | 1 μl |
| Mai hana RNase (40U/ul) | 0.5 μl |
| RTL Reverse Transcriptase 2.0 (15U/ul) | 0.5 μl |
| 10 × HC RTL Buffer | 2 μl |
| Ruwa marar Nukiliya | Har zuwa 20 μl |
Bayanan kula:
1) Adadin da aka ba da shawarar na Total RNA shine 1ng ~ 1μg
2) Adadin da aka ba da shawarar na mRNA shine 50ng ~ 100ng
Thermo-Yanayin hawan keke na yau da kullun amsa:
| Zazzabi (°C) | Lokaci |
| 25 °Ca | 5 min |
| 55 °C | Minti 10b |
| 80 °C | Minti 10 |
Bayanan kula:
1) Idan aka yi amfani da Random Primer Mix, matakin shiryawa a 25°C.
2) Idan an yi amfani da mahaɗin farko na manufa, matakin shiryawa a 55 ° C na 10 ~ 30mins.
RT-LAMP Protocol
| Abubuwan da aka gyara | Ƙarar | Ƙarshe Tattaunawa |
| Samfura RNA | na zaɓi | ≥10 kwafi |
| dNTP Mix (10mM) | 3.5 ml | 1.4 mm |
| Fitilar FIP/BIP (25×) | 1 μl | 1.6 m |
| F3/B3 Fim (25×) | 1 μl | 0.2 μM |
| LoopF/Madauki na Farko (25×) | 1 μl | 0.4 μM |
| Mai hana RNase (40U/μL) | 0.5 μl | 20 U/Reaction |
| RTL Reverse Transcriptase 2.0 (15U/μL) | 0.5 μl | 7.5 U/ amsawa |
| Bst V2 DNA Polymerase (8U/μL) | 1 μl | 8 U/Aiki |
| MgSO4 (100mM) | 1.5 μl | 6 mM (Jimlar 8mM) |
| 10×HC RTL Buffer (ko 10×HC Bst V2 Buffer) | 2.5 ml | 1 × (2mM mg2+) |
| Ruwa marar Nukiliya | Har zuwa 25 μl | - |
Bayanan kula:
1) Mix ta hanyar vortexing da centrifuge a taƙaice don tattarawa.Yawan zafin jiki na yau da kullun a 65 ° C na awa 1.
2) Abubuwan buffer guda biyu suna yin aiki tare kuma suna da abun da ke ciki iri ɗaya.
Bayanan kula
1.Wannan samfurin zai samar da wani farin m lokacin da aka adana a -20 °C.Cire shi daga -20 ° C kuma sanya shi a kan kankara na kimanin minti 10.Bayan narkewa, ana iya amfani dashi ta hanyar girgiza da haɗuwa.
2.A cDNA samfurin za a iya adana a -20 ° C ko -80 ° C ko amfani da nan da nan don PCR dauki.
3.Don hana gurɓatar RNase, da fatan za a kiyaye yankin gwaji mai tsabta, kuma sanya safofin hannu masu tsabta da abin rufe fuska yayin aiki.
