 |
|
 |
|
|
| | | | M-MuLV Reverse Transcriptase | | | |
|
|
M0253L |
|
50,000 units
|
|
200,000 units/ml |
|
|
|
M0253S |
|
10,000 units
|
|
200,000 units/ml |
|
- Isolated from a recombinant source
- Synthesizes cDNA from single-stranded RNA or DNA
Description:
Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase is an RNA-directed DNA polymerase. This enzyme can synthesize a complementary DNA strand initiating from a primer using either RNA (cDNA synthesis) or single-stranded DNA as a template. (1-4). M-MuLV Reverse Transcriptase lacks 3´ → 5´ exonuclease activity.
Source:
The gene encoding M-MuLV Reverse Transcriptase is expressed in E.coli in a vector that results in 16 additional amino acids at the N-terminus and 13 amino acids at the C-terminus. This construct results in a fully functional Reverse Transcriptase protein with a functional RNase H domain.
Reagents Supplied:
M-MuLV Reverse Transcriptase Reaction Buffer (10X)
Enzyme Properties
3´ to 5´ Exonuclease: No
5´ to 3´ Exonuclease: No
Strand Displacement: Yes
Heat Inactivation:
65°C for 20 minutes
Molecular Weight:
Theoretical: 78,000 daltons
Specific Activity:
500,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X M-MuLV Reverse Transcriptase Reaction Buffer
Supplemented with 100 μM dNTPs (not included)
Incubate at
37°C.
1X M-MuLV Reverse Transcriptase Reaction Buffer:
50 mM Tris-HCl
75 mM KCl
3 mM MgCl2
10 mM Dithiothreitol
pH 8.3 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme that will incorporate 1 nmol of dTTP into acid-insoluble material in a total reaction volume of 50 μl in 10 minutes at 37°C using poly(rA).oligo(dT) as template primer with 50 mM Tris-HCI (pH 8.3), 6 mM MgCl2, 10 mM dithiothreitol, 0.5 mM [3H]-dTTP and 0.4 mM poly(rA).oligo(dT)12-18.
Concentration:
200,000 units/ml
Storage Conditions:
50 mM Tris-HCl
150 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
0.1% NP-40
pH 7.6 @ 25°C
Storage Temperature:
-20°C
FAQs
- Can M-MuLV Reverse Transcriptase be used in other NEBuffers?
- Does M-MuLV Reverse Transcriptase require DTT?
- Are NEB DNA Polymerases supplied with dNTPs?
- How can the length of the product generated by M-MuLV Reverse Transcriptase be increased?
- How can the yield be improved when using M-MuLV Reverse Transcriptase?
- What are some of the reasons for M-MuLV Reverse Transcriptase reaction failure?
- Is M-MuLV Reverse Transcriptase active at temperatures higher than 42°C?
- Is M-MuLV Reverse Transcriptase used in the Protoscript First Strand cDNA Synthesis Kit?
- Can DNA be used as a template for M-MuLV Reverse Transcriptase?
- Does NEB carry M-MulV Reverse Transcriptase RnaseH- (NEB# M0248)?
Protocols
Protocols for M-MuLV Reverse Transcriptase
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
M-MuLV Reverse Transcriptase is tested for its ability to synthesize full length cDNAs from crude or purified RNA templates. Purified free of detectable levels of RNase, endonuclease and exonuclease activities.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of ΦX174 DNA
and 100 units of M-MuLV Reverse Transcriptase incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of M-MuLV Reverse Transcriptase with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.2% of the total radioactivity.
RNase Assay:
Incubation of a 50 μl reaction containing 100 units of M-MuLV Reverse Transcriptase with 1
μg of MS2 RNA for 1 hour at 37ºC
resulted in no detectable degradation of the RNA as determined by agarose gel electrophoresis.
References
- Verma, I.M. (1975) J. Virol., 15, 843-854.
- Gerard, G.F. and Grandgenett, D.P. (1975) J. Virol., 15, 785-797.
- Roth, M.J., Tanese, N. and Goff, S.P. (1985) J. Biol. Chem., 260, 9326-9335.
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd Ed.), 5.52-5.55, 8.11-8.17.
Reagents Sold Separately
M-MuLV Reverse Transcriptase Reaction Buffer
Companion Products
ProtoScript® First Strand cDNA Synthesis Kit
|
|
|
|
|
