M-MLV Neoscript Reverse Transcriptase

Neoscript Reverse Transcriptase is a reverse transcriptase inowanikwa ne mutation screening yeM-MLV gene yeMoloney murine leukemia virus mabviro uye kutaura muE.coli.Iyo enzyme inobvisa RNase H chiitiko, ine yakakwira tembiricha kushivirira, uye inokodzera yakakwira-tembiricha reverse transcription.Naizvozvo, zvinobatsira pakubvisa mhedzisiro isingafadzi yeRNA yepamusoro-yepamusoro dhizaini uye isiri-chaiyo zvinhu pane cDNA synthesis, uye ine kugadzikana kwepamusoro uye reverse transcription synthesis kugona.Iyo enzyme ine yakakwirira kugadzikana uye reverse transcription synthesis kugona.

Zvikamu

1.200 U/μL Neoscript Reverse Transcriptase

2.5 × Yekutanga-Strand Buffer (kusarudza)

* 5 × Yekutanga-Strand Buffer haina dNTP, ndapota wedzera dNTPs paunenge uchigadzirira maitiro

Recommended Application

1.Imwe-nhanho qRT-PCR.

2.Kuonekwa kwehutachiona hweRNA.

Storage Condition

-20°C kuitira kuchengetedza kwenguva refu, inofanira kusanganiswa zvakanaka isati yashandiswa, dzivisa kugaro chando-nyorova.

Chikamu Tsanangudzo

Chimwe chikamu chinosanganisa 1 nmol yedTTP mumaminetsi gumi pa37°C uchishandisa poly(A)•oligo(dT)₂₅se template/primer.

Quality Control

1.SDS-PAGE electrophoretic kuchena kukuru kupfuura 98%.

2.Amplification senitivity, batch-to-batch control, kugadzikana.

3.Hapana exogenous nuclease chiitiko, hapana exogenous endonuclease kana exonuclease kusvibiswa.

Reaction Setup yeFirst Chain Reaction Solution

1.Kugadzirira kwemusanganiswa wekuita

Notes:a/b: Ndokumbira usarudze mhando dzakasiyana dzemaprimers zvinoenderana nezvido zvako zvekuyedza.

2.Pisa pa65°C kwe5mins uye wotonhorera nekukurumidza paaizi kwema2mins.

3.Wedzera zvinotevera zvikamu kune iri pamusoro system kune yakazara vhoriyamu ye20µL uye sanganisa zvinyoro nyoro:

4.Ndapota ita mhinduro maererano nemamiriro anotevera:

(1) Kana Random Primer ikashandiswa, maitiro anofanirwa kuitwa pa25 ℃ kwe10mins, uyezve pa50 ℃ ye30 ~ 60mins;

(2) Kana Oligo dT kana chaiyo primers yakashandiswa, maitiro anofanirwa kuitwa pa50 ℃ kwe30 ~ 60mins.

5.Kupisa pa95 ℃ kwe5mins kudzima Neoscript Reverse Transcriptase uye kumisa kuita.

6.Reverse transcription products zvinogona kushandiswa zvakananga muPCR reaction uye fluorescence quantitative PCR reaction, kana kuchengetwa pa -20 ℃ kwenguva yakareba.

PCR Rchiito:

1.Kugadzirira kwemusanganiswa wekuita

Notes:a: Kana yakawandisa yekutanga chain reaction solution yakawedzerwa, PCR reaction inogona kudziviswa.

2.PCR Reaction Procedure

Notes

1.Inokodzera reverse transcription tembiricha optimization mumhando ye42 ℃ ~ 55 ℃.

2.Iyo ine kugadzikana kuri nani, yakakodzera kune yakakwira tembiricha reverse transcription amplification.Pamusoro pezvo, yakanakira kupfuura nemumatunhu akaoma eRNA.Uyezve, izvozvoinokodzera nhanho imwe-multiplex fluorescence quantitative RT-PCR yekuona.

3.Kuenderana kwakanaka neakasiyana PCR amplification enzymes uye inokodzera yakakwira senitivity RT-PCR maitiro.

4.Inokodzera yakakwirira senitivity imwe-nhanho fluorescence kuwanda RT-PCR maitiro, zvinobudirira kuvandudza mwero wekuona wekushomeka kwematemplate.

5.Inokodzera cDNA raibhurari kuvaka.