Excellgen/ExcellScript Thermostable Reverse Transcriptase/EG-1029/4,000,000 Units
| Description | ExcellScript Thermostable M-MuLV Reverse Transcriptase is a DNA polymerase which utilizes RNA as a substrate and exhibits no measurable proofreading 3′→5′ exonuclease function. This thermostable reverse transcriptase can perform cDNA synthesis reactions throughout a wide range of temperatures from 42°C to 72°C. |
| Applications | - First strand cDNA synthesis for RT-PCR and real-time RT-PCR
- Synthesis of cDNA for cloning and expression
- Generation of labeled cDNA probes for microarrays
- DNA labeling
- Analysis of RNA by primer extension
- Isothermal nucleic acid amplification such as LAMP
|
| Source | A recombinant E. coli strain carrying an engineered Moloney-Murine Leukemia Virus Reverse Transcriptase gene |
| Quality Control | - The absence of endo-, exodeoxyribonucleases, phosphatases, and ribonucleases confirmed by appropriate quality tests.
- Functionally tested in first strand cDNA synthesis.
|
| Unit definition | 1 unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid insoluble material in 10 minutes at 37°C using poly r(A)/oligo (dT) as a substrate. |
| Components | - Thermostable M-MuLV Reverse Transcriptase: 200 units/µL in 50 mM Tris-HCl, 150 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% NP-40, 50% glycerol, pH 7.6 @ 25°C
- 10X Reaction Buffer: 500 mM Tris-HCl, 750 mM KCl, 30 mM MgCl2, 100 mM DTT pH 8.3 @ 25°C
|
| Protocol | Primer Annealing: Combine the following in an RNase-free reaction vessel:| Amount | Description | Final Concentration | | 1 µL | 25 mM dNTP Solution (N2050L | 2.0 mM | | X µL | 1 ng-2 µg Total RNA -or- | | | X µL | 5-500 ng mRNA (polyA selected) | | | 1 µL | Oligo (dT)12-18 (500 µg/ml) -or- | 40 µg/mL | | 1 µL | Random Primers (125 µg/ml) -or | 10 µg/mL | | 1 µL | GSP Primer (2 pmol) | 165 µM | | X µL | Sterile Water | N/A | | 10 µL | Total Volume |
Heat reaction for 5 minutes at 65°C. Spin briefly (5 sec) to pull down condensate and place immediately on ice.Add 1 µL 10X M-MuLV RT Buffer and DNase-free water to a final volume of 10 µL per reaction.Incubate:- If using Oligo (dT) or GSP primers: 2 minutes @ 42°C
- If using Random primers: 2 minutes @ 25°C
Add 1 µL (200 units) thermostable M-MuLV Reverse Transcriptase and mix by gently pipetting sample. (Note: if using random primers, pre-incubate reaction @25°C for 10 minutes).Incubate at 42 ~ 72 °C for 45-60 minutes.Inactivate enzyme at 95°C for 10 minutes.Store products at -20°C or proceed to next step. |
| Shipping | 4°C |
| Note | For research use only |
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