M-MLV Reverse Transcriptase (RNaseH-)

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 in: 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°C using poly(A)•oligo(dT)25 as template-primer.

Quality Control Assays

• Endonuclease Activity: Incubation of 400 U of 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 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 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:

A:

Thermocycling Conditions for a routine reaction：

ⓐ 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.