M-MLV Reverse Transcriptase (RNase H-)


Product information

Product NameCat. No.Size
HMD3301S20 KU
M-MLV Reverse Transcriptase (RNaseH-)HMD3301M80 KU
HMD3301L200 KU


Description:

M-MLV ( Moloney Murine Leukemia Virus ) reverse transcriptase (RNaseH- )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. M-MLV reverse transcriptase lacks 3´ → 5´ exonuclease activity and RNase H activity.

Storage and transportation:

Storage at -20 ℃ and transportation≤0℃

Supplied :

50 mM Tris-HCl(pH 7.6 at 25℃), 150 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% IGEPAL CA-630 and 50% Glycerol.

Unit definition:

One unit incorporates 1 nmol of dTTP into acid-precipitable material in 10 minutes at 37°Cusing poly(A)•oligo(dT)25 as template primer.

Quality Control Assays

  • Endonuclease Activity: Incubation of 400 U of an enzyme with 4 μg pUC19 DNA for 4 hours at 37°C resulted in no detectable degradation of the DNA as determined by gel electrophoresis.
  • Exonuclease Activity: Incubation of a 50 µL reaction containing a minimum of 500 U of an enzyme with 10 nM 5´-FAM oligonucleotide for 30 minutes at 37℃ yields no detectable degradation.
  • RNase Activity: Incubation of a 10 µL reaction containing 400 U of an enzyme with 1μg of MS2 RNA transcripts for 1 hour at 37°C resulted in no detectable degradation of the RNA as determined by gel electrophoresis.

Reaction setup:

ComponentVolume
Template RNAoptional
Oligo(dT)23(50 μM or Random Primer Mix(60μM)2 μL
dNTP Mix (10 mM each)1 μL
RNase Inhibitor(40 U/μL)0.2 μL
M-MLV Reverse Transcriptase (200 U/μL)1 μL
10 x MMLV Buffer2 μL
Nuclease-free WaterUp to 20 μL

a:

Total RNA1 ng – 1 μg
Poly(A) RNA50 pg – 100 ng

Thermocycling Conditions for a routine reaction:

TemperatureTime
25 ℃Ⓓ10 min
42 – 55℃Ⓐ1 h
95 ℃5 min

Ⓓ If Random Primer Mix is used, an incubation step at 25℃ before 42 ℃. ⒶIf the target primer mix is used, an incubation step at 42 – 55℃ for 1h.

The Certificate of Analysis (COA) is a signed document that includes the storage temperature

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