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M-MLV RTase (M-MLV Reverse Transcriptase)

Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase is an RNA-dependent DNA polymerase that synthesizes the first strand of complementary cDNA from a single-stranded RNA template with hybridized primer. This kit features high activity formulation of M-MLV RT and 5X reverse transcription buffer which are capable of full-length cDNA synthesis and high cDNA yields.
No. Size Price Qty Status
C15021-20000U 20,000U $128.00 Inquiry
C15021-50000U 50,000U $296.00 Inquiry
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Package & Component :
Cat. Name Amount
C15021-20000U M-MLV Reverse Transcriptase (200 U/μL) 20,000 U
5X M-MLV Reverse Transcriptase Reaction Buffer 1 mL
C15021-50000U M-MLV Reverse Transcriptase (200 U/μL) 50,000 U
5X M-MLV Reverse Transcriptase Reaction Buffer 1 mL

Source:
Escherichia coli

Purity:
>98% as determined by SDS-PAGE (purified by Ni-NTA chromatography).

Unit Definition:
One unit is defined as the amount of the enzyme incorporates 1 nmol of dTTP into acid-insoluble product in 10 minutes at 37°C.

Reaction Condition:
1X M-MLV Reverse Transcriptase Buffer, supplemented with dNTP mix, template and primer, Incubate at 37°C for synthesis of first strand cDNA.
5X M-MLV Reverse Transcriptase Buffer: 250 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 375 mM KCl, and 50 mM DTT.

Storage Buffer:
M-MLV Reverse Transcriptase is supplied in 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 0.25 mM EDTA, 0.01% NP-40 (v/v), 2.5 mM DTT, 50% glycerol (v/v).

Storage:
Stored at -20°C. Avoid repeated freeze/thaw cycles.

Notes:
After the reaction is complete, M-MLV RTase can be inactivated by incubation at 65°C for 20 minutes.
First strand cDNA synthesis:
1. Place all required reagents to a nuclease-free microcentrifuge tube and following the order suggested below.
Component Amount Final concentration
Oligo (dT)12-18 (50 μM) or random primer mix (60 μM) 1 μL -
Total RNA template X μL 1 μg
Nuclease-Free H2O Y μL -
Total reaction volume 13.5 μL -
2. Heat the tube to 65°C for 10 minutes to denature the secondary structure within RNA template. Immediately cool the tube on ice for 1 minute and centrifuge briefly in microcentrifuge. Add the following components to the annealed primer/RNA template, prepare on ice.
Component Amount Final concentration
5X Reverse Transcriptase Reaction Buffer 4 μL 1X
10 mM dNTPs (each) 1 μL 0.5 mM each
RNase Inhibitor X μL 20 U/rxn
M-MLV RTase 1 μL 200 U/rxn
Nuclease-Free H2O Y μL -
Total reaction volume 20 μL -
3. Incubate at 37°C for 1 hour. The extension temperature may be adjusted from 37°C to 42°C.
4. Inactivate the reaction at 65°C for 20 minutes. The cDNA products should be store at -20°C.
5. Reaction preparations may be scaled up or down proportionately.