M-MLV Reverse Transcriptase M2 HYA212
M-MLV Reverse Transcriptase M2 is a reverse transcriptase obtained by mutation screening of the M-MLV gene from Moloney murine leukemia virus and expression in E. coli. The enzyme has RNase H activity removed and has higher temperature tolerance. It is suitable for high-temperature reverse transcription, which is conducive to eliminating the adverse effects of RNA high-level structure and non-specific factors on cDNA synthesis. It has higher stability and reverse transcription synthesis ability.
Components
1. 200 U/µL M-MLV Reverse Transcriptase M2
2. 5×M-MLV Reverse Transcriptase Buffer (optional)
*5×M-MLV Reverse Transcriptase Buffer does not contain dNTPs. Please add dNTPs when preparing the reaction system.
Storage
Store at -25°C~ -15°C.
Unit Definition
1 activity unit (U) is defined as the amount of enzyme required to convert 1 nmol of dTTP into acid-insoluble substances at 37°C for 10 min using Poly (rA)·Oligo (dT) as template/primer.
Quality Control
1. Purity >98% as determined by SDS-PAGE electrophoresis.
2. Amplification sensitivity, batch-to-batch variation, and stability.
3. Free of exogenous nuclease activity; no contamination by exogenous endonucleases or exonucleases.
Instructions
1. Refer to the table below to prepare the system
|
Components |
Volume |
|
Template RNA |
total RNA ≤ 5 µg；mRNA ≤ 1 µg |
|
Oligo(dT)12-18 Primer* |
50 pmol |
|
Or Random Primer * |
50 pmol（20~100 pmol） |
|
Or Gene specific Primer * |
2 pmol |
|
dNTPs（10 mM each） |
1 μL |
|
Nuclease-free Water |
Up to 10 μL |
*Choose different types of primers according to experimental needs.
2. Heat at 65℃ for 5 min, and quickly cool on ice for 2 min;
3. Add the following components to the above system to a total volume of 20 µL, and mix gently.
|
Components |
Volume |
|
5×M-MLV Reverse Transcriptase Buffer |
4 uL |
|
200 U/µL M-MLV Reverse Transcriptase M2 |
1 uL |
|
40 U/µL RNase Inhibitor |
1 uL |
|
Nuclease-free Water |
Up to 20 μL |
4. Perform the reaction under the following conditions.
1) If using Random Primer, incubate at 25℃ for 10 min first; then incubate at 50℃ for 30~60 min;
2) If using Oligo dT or specific primers, incubate at 50℃ for 30~60 min.
5. Inactivate M-MLV Reverse Transcriptase by heating at 95℃ for 5 min and terminate the reaction.
6. The reverse transcription product can be used directly for PCR reaction and fluorescence quantitative PCR reaction, or stored at -20℃ for a long time.
Technical Description
1. Suitable for reverse transcription temperature optimization in the range of 42℃~55℃.
2. With better stability, it is suitable for high-temperature reverse transcription amplification, which is conducive to effectively passing through the complex structure area of RNA and suitable for one-step multiplex fluorescence quantitative RT-PCR detection.
3. It has good compatibility with various PCR amplification enzymes and is suitable for high-sensitivity RT-PCR reactions.
4. It is suitable for high-sensitivity one-step fluorescence quantitative RT-PCR reactions, effectively improving the detection rate of low-concentration templates.
5. It is suitable for cDNA library construction.
6. 3’and 5’RACE.