Store at -25 ~ -15℃ for 2 years
[1] Roth, M J , N. Tanese , and S. P. Goff . "Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. " Journal of Biological Chemistry 260.16(1985):9326.
[2] Kotewicz, Michael L , et al. "Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity." Nucleic Acids Research 16.1(1988):265-277.
Component | UA070078-10 Rxns | UA070078-50 Rxns |
DNase I, RNase-free (2 U/μL) | 10 μl | 50 μl |
10X Reaction Buffer with MgCl2 for DNase I | 10 μl | 50 μl |
50 mM EDTA | 10 μl | 50 μl |
M-MLV(H-) Reverse Transcriptase (200 U/μl) | 10 μl | 50 μl |
5× Reaction Buffer | 40 μl | 200 μl |
RNase Inhibitor (40 U/μl) | 5 μl | 25 μl |
dNTP Mixture (10 mM each) | 10 μl | 50 μl |
Oligo(dT)18 Primer (50 μM) | 10 μl | 50 μl |
Random Hexamer Primer (50 μM) | 20 μl | 100 μl |
RNase Free dH2O | 200 μl | 1 ml |
I. Removal of genomic DNA from RNA preparations
1. The reaction system is formulated as follows:
Component | Volume |
RNA | 1 μg |
10X Reaction Buffer with MgCl2 | 1 μl |
DNase I, RNase-free (2 U/μL) | 1 μl |
RNase Free dH2O | To 10 μl |
2. Incubate at 37 °C for 10 min.
3. Add 1 μL 50 mM EDTA and incubate at 65 °C for 10 min. RNA hydrolyzes during heating with divalent cations in the absence of a chelating agent.
4. Use the prepared RNA as a template for reverse transcriptase.
II. First Strand cDNA Synthesis
1. Set up the reverse transcription reaction according to the following table
2. Optional. If the RNA template is GC-rich or contains secondary structures, mix gently, centrifuge briefly and incubate at 65 °C for 5 min. Chill on ice, spin down and place the vial back on ice.
3. Add the following components in the indicated order:
Component | Volume |
Reaction Buffer (5X) | 4 μl |
RNase Inhibitor (40 U/μl) | 0.5 μl |
dNTP Mix (10mM each) | 2 μl |
M-MLV(H-) Reverse Transcriptase (200 U/μl) | 1 μl |
Total Volume | 20 μl |
4. Mix gently (gently with a pipette or gently with a vortex mixer at the lowest speed), then centrifuge the precipitated liquid.
5. If Oligo(dT)18 or gene-specific primers are used, incubate at 42℃ for 10-60 min. If random hexamer(random hexamer) is used as primer, incubate at 25℃ for 10min, and then incubate at 42℃ for 10-60 min. Reverse transcription of cDNA below 3 kb can be done for 10min, reverse transcription of 3-6 kb cDNA can be done for 30 min, and reverse transcription of cDNA above 6 kb is recommended for 60 min. When random hexamer(random hexamer) is used as primer and subsequently used for qPCR, 10min is sufficient for subsequent detection of genes of any length.
6. Terminate the reaction by heating at 70 °C for 5 min. The reverse transcription reaction product can be directly used in PCR applications or stored at -20 °C for less than one week. For longer storage, -70 °C is recommended.
III. PCR Amplification of First Strand cDNA
The product of the first strand cDNA synthesis can be used directly in PCR or qPCR. The volume of first strand cDNA synthesis reaction mixture should not comprise more than 1/10 of the total PCR reaction volume. Normally, 2 μL of the first strand cDNA synthesis reaction mixture is used as template for subsequent PCR in 50 μL total volume.
1. Use DEPC to dispose of all equipment used in the study, or purchase equipment that has been proved to be nucleic acid-free. Wear gloves during the study and change them frequently to avoid RNA enzyme contamination.
2. Ensure that there is no RNA enzyme contamination in the reagents used.
3. Keep the kit tightly sealed. During the reverse transcription process, all tubes must be securely fastened.
4. Purified RNA must be free of salt, metal ions, ethanol and phenol, which will interfere with the first strand cDNA synthesis reaction. Trace contaminants can be removed by precipitation of RNA with ethanol.
5. In order to ensure the effective retrotranscriptional reaction, high-quality RNA templates need to be used.
6. When the template content is low or the subsequent PCR amplification fragment is too long, the reverse transcription time can be appropriately extended.
Genomic DNA in 2 µg total RNA extracted from HEK293 cells was removed by DNase I and then reverse-transcribed in a 20 µL reverse transcription system. After reverse transcription, 2 μl of the reverse transcription product was obtained, and the β_Actin cDNA 197bp fragment was amplified by qPCR and detected by electrophoresis. The following is the effect diagram of qPCR detection after reverse transcription of total RNA.
Lane 1 Negative Control-1 (Only qPCR negative controls without added DNA template);
Lane 2 Negative Control-2 (Total RNA was not treated with DNase I and only reverse transcriptase was not added);
Lane 3 Negative Control-3 (Total RNA was treated with DNase I and only reverse transcriptase was not added);
Lane 4 UA M-MLV(H-) Reverse Transcriptase 200U;
Lane 5 Competing Product N 200U;
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