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Biosan LLC is an R&D executor with the support of the Foundation for Assistance to Small Innovative Enterprises.Reverse transcriptase M-MuLV
Features: Removal of the RNase H activity resulted in an increase of full-length cDNA products.
M-MLV Reverse Transcriptase (RNase H Minus)
Description: Purified from E. coli strain carrying a plasmid with the cloned portion of the pol gene encoding Moloney Murine Leukemia Virus Reverse Transcriptase. The enzyme possesses RNA- and DNA-dependent DNA polymerase activity, but lacks ribonuclease H activity.
Unit definition: One unit is the amount of the enzyme incorporating 1 nmol of dTTP into acid-insoluble form in 10 min at 37ºС in 50 mM Tris-HCl, pH 8.3, 40 mM KCl, 5 mM DTT, 6 mM MgCl2, poly A 2 A260/ml, 2 uM d(pT)12, 50 uM [H3]dTTP, 100 ug/ml BSA.
Storage conditions: 50 mM Tris-HCl, pH 8.0 (at 25 ºС), 100 mM NaCl, 1 mМ EDTA, 5 mМ DTT, 50 % glycerol, 0,1 % NP-40.
Store at –20 °C.
Quality control: Tested for the absence of exo-, endodeoxyribonuclease and ribonuclease activity.
Applications:
v Production of templates for PCR
v First strand cDNA synthesis
v Construction of cDNA libraries
v Labeling and filling-in 3’-end-termini
v mRNA 5’-end mapping by primer extension analysis
v RNA and DNA sequencing
Protocol: cDNA synthesis with M–MuLV Reverse transcriptase
10 pmole of strand-specific primer (or 250-500ng of oligo-dT for each μg of RNA)
add water up to 8 μl
2. Incubate the mixture 10 min at 70°C, then 10-15min at room temperature (for the specific primer) or place in ice in
the case of oligodT or random primer
3. Add into the mixture:
- 4 μl of 5xRT buffer (250mM TrisHCl, pH8,3; 500mM KCl, 15mM MgCl2)
- 2 μl of 0,1M DTT
- 1 μl of dNTP mix (10mM of each dNTP; Cat.-No: 110001 and 110002)
- RNAsin – 20-40 units (optional)
- Revertase – 200 units
- H2O – up to 20 μl
4. Incubate the mixture at 37-55°C during 30 – 120 min. The time of reaction depends on the length of cDNA, 30 min is enough for cDNA in range of 500 bp in length, 120 min is suitable for cDNA more then 1,5 kb. The temperature of the reaction depends on the structural features of RNA. Use increased temperature (up to 55°C) for the highly structured RNA. The optimal temperature and reaction time should be adjusted for each particular RNA. We recommend to use buffer with pH 8.8 if the reaction is performed at elevated temperature.
5. Heat the mixture 10 min at 65-70°C to inactivate the Revertase.
6. Use the mixture for PCR or for other application.
Revertase is free from RNase activities, meanwhile we recommend to add RNasin into the mixture to inhibit possible RNase contaminations of the sample.
Description: Purified from E. coli strain carrying a plasmid with the cloned portion of the pol gene encoding Moloney Murine Leukemia Virus Reverse Transcriptase. The enzyme possesses RNA- and DNA-dependent DNA polymerase activity, but lacks ribonuclease H activity.
Unit definition: One unit is the amount of the enzyme incorporating 1 nmol of dTTP into acid-insoluble form in 10 min at 37ºС in 50 mM Tris-HCl, pH 8.3, 40 mM KCl, 5 mM DTT, 6 mM MgCl2, poly A 2 A260/ml, 2 uM d(pT)12, 50 uM [H3]dTTP, 100 ug/ml BSA.
Storage conditions: 50 mM Tris-HCl, pH 8.0 (at 25 ºС), 100 mM NaCl, 1 mМ EDTA, 5 mМ DTT, 50 % glycerol, 0,1 % NP-40.
Store at –20 °C.
Quality control: Tested for the absence of exo-, endodeoxyribonuclease and ribonuclease activity.
Applications:
v Production of templates for PCR
v First strand cDNA synthesis
v Construction of cDNA libraries
v Labeling and filling-in 3’-end-termini
v mRNA 5’-end mapping by primer extension analysis
v RNA and DNA sequencing
Protocol: cDNA synthesis with M–MuLV Reverse transcriptase
- Mix in the tube
10 pmole of strand-specific primer (or 250-500ng of oligo-dT for each μg of RNA)
add water up to 8 μl
2. Incubate the mixture 10 min at 70°C, then 10-15min at room temperature (for the specific primer) or place in ice in
the case of oligodT or random primer
3. Add into the mixture:
- 4 μl of 5xRT buffer (250mM TrisHCl, pH8,3; 500mM KCl, 15mM MgCl2)
- 2 μl of 0,1M DTT
- 1 μl of dNTP mix (10mM of each dNTP; Cat.-No: 110001 and 110002)
- RNAsin – 20-40 units (optional)
- Revertase – 200 units
- H2O – up to 20 μl
4. Incubate the mixture at 37-55°C during 30 – 120 min. The time of reaction depends on the length of cDNA, 30 min is enough for cDNA in range of 500 bp in length, 120 min is suitable for cDNA more then 1,5 kb. The temperature of the reaction depends on the structural features of RNA. Use increased temperature (up to 55°C) for the highly structured RNA. The optimal temperature and reaction time should be adjusted for each particular RNA. We recommend to use buffer with pH 8.8 if the reaction is performed at elevated temperature.
5. Heat the mixture 10 min at 65-70°C to inactivate the Revertase.
6. Use the mixture for PCR or for other application.
Revertase is free from RNase activities, meanwhile we recommend to add RNasin into the mixture to inhibit possible RNase contaminations of the sample.
