M-MLV Reverse Transscriptase
RevScript Reverse transcriptase yog tau los ntawm genetic engineering technology.Nws muaj ntau dua cDNA synthesis muaj peev xwm, thermal stability thiab cov tshuaj tiv thaiv kub txwv (txog 60 ° C).Cov khoom sib txuas cDNA yog txog 10 kb.Nws txhim kho kev sib raug zoo ntawm cov qauv thiab tsim nyog rau kev rov qab hloov pauv ntawm RNA cov qauv nrog cov qauv theem nrab lossis cov noob qes qis.
Cheebtsam
| Cheebtsam | HC2003 B (10,000 U) | HC2003 B (5 * 10,000U) | HC2003 B (200,000 U) |
| RevScript Reverse Transcriptase (200U / μL) | 50l ua | 5 × 50 hli | 1 ml ib |
| 5 × RevScript Buffer | 250 mL | 1.25 ml yog ' | 5 ml ib |
Kev cia khoom
Cov khoom no yuav tsum khaws cia ntawm -25 ° C ~ -15 ° C rau 2 xyoos.
Unit txhais
Ib chav tsev suav nrog 1 nmol ntawm dTTP rau hauv cov khoom siv acid-insoluble hauv 10 feeb ntawm 37 ° C siv Oligo (dT) ua primers.
Kev teeb tsa kev daws teeb meem
1.Denaturation ntawm RNA template (Cov kauj ruam no yog xaiv tau, denaturation ntawm RNA template pab qhib cov txheej txheem thib ob, uas yuav txhim kho cov txiaj ntsig ntawm thawj strand cDNA.)
| Cheebtsam | Ntim (μL) |
| RNase dawb ddH2O | Rau 13 |
| Oligo (dT)18 (50 μmol / L) los yog Random Primer (50 μmol / L) Los yog Gene Specific Primers (2 μmol / L) | 1 |
| lub 1 | |
| lub 1 | |
| RNA template | X a |
Nco tseg:
1) a: Tag Nrho RNA: 1-5 ug lossis mRNA: 1-500 ng
2) Ua kom sov ntawm 65 ° C rau 5 feeb, tom qab ntawd hloov mus rau dej khov tam sim kom txias rau 2 feeb.Luv luv centrifugation los sau cov tshuaj tiv thaiv kua, ntxiv cov tshuaj tiv thaiv rov qab los ntawm cov tshuaj raws li qhia hauv qab no.Maj mam pipette sib tov.
1.Kev npaj cov tshuaj tiv thaiv sib tov (20 μL ntim)
| Cheebtsam | Ntim (μL) |
| Kev sib xyaw ntawm cov kauj ruam dhau los | 13 |
| 5 × Tsis muaj | 4 |
| dNTP sib tov (10nmol / L) | 1 |
| Rov qab Transscriptase (200 U / μL) | 1 |
| RNase inhibitor (40 U / μL) | 1 |
1.Ua cov tshuaj tiv thaiv raws li hauv qab no:
| Qhov kub thiab txias (°C) | Sijhawm |
| 25 ° Ca | 5 feeb |
| 42 ° Cb | 15-30 feeb |
| 85 ° Cc | 5 feeb |
Nco tseg:
1) a.Incubating ntawm 25 ° C rau 5mins tsuas yog yuav tsum tau siv cov random hexamers.Thov hla cov kauj ruam no thaum siv Oligo (dT)18los yog Gene Specific Primer.
2) ib.Qhov pom zoo rov qab hloov pauv kub yog 42 ° C, Rau cov qauv uas muaj cov txheej txheem thib ob nyuaj lossis cov ntsiab lus GC siab, nws raug nquahu kom nce qhov ntsuas kub rau 50-55 ° C.
3) c.Tshav kub ntawm 85 ° C rau 5mins kom inactivate reverse transcriptase.
4) Cov khoom tuaj yeem siv ncaj qha rau hauv PCR lossis qPCR cov tshuaj tiv thaiv, lossis khaws cia ntawm -20 ° C rau lub sijhawm luv luv.Nws raug nquahu kom aliguot cov khoom thiab khaws cia ntawm -80 ° C rau kev khaws cia ntev.Tsis txhob nquag khov-thaw.
5) Cov khoom tsim nyog rau ib-kauj ruam RT-qPCR, nws raug nquahu kom ntxiv 10-20 U thim rov qab transcriptase rau txhua txhua 25μL cov tshuaj tiv thaiv kab mob, lossis maj mam nce tus nqi rov qab transcriptase raws li qhov xwm txheej tiag tiag.
Sau ntawv
1.Thov khaws qhov chaw sim kom huv si;Cov hnab looj tes huv thiab qhov ncauj qhov ntswg yuav tsum hnav thaum ua haujlwm.Tag nrho cov khoom siv uas siv hauv qhov kev sim yuav tsum yog RNase dawb los tiv thaiv RNase paug.
2.Txhua cov txheej txheem yuav tsum tau ua ntawm cov dej khov kom tiv thaiv RNA degradation.
3.Cov qauv zoo RNA raug pom zoo los ua kom muaj kev ua haujlwm siab ntawm kev rov qab rov qab.
4.Cov khoom no tsuas yog siv rau kev tshawb nrhiav.
5.Thov ua haujlwm nrog cov tsho loj thiab cov hnab looj tes pov tseg, kom koj muaj kev nyab xeeb.
