Manual

Product Number: RT01

Storage condition

-20°C

Component

MMLV (RNase H-) (200U/uL)

5×RT Buffer (with DTT)

Description

         MMLV Reverse Transcriptase isolated from E. coli carrying rat leukemia virus pol gene consists of a single peptide, molecule weight 71kd. MMLV Reverse Transcriptase, RNase H Minus is an RNA-dependent DNA polymerase with no detectable RNase H activity. A point mutation in the RNase H domain increases the thermostability of the enzyme and support greater cDNA yield of full-length transcripts than wild type M-MLV Reverse Transcriptase.

Features

         Weak RNase H activity

         High cDNA yield

Application

Synthesis of the first chain cDNA Library construction one-step RT-PCR primer extension 3′ and 5′ RACE.

Source

Recombination of E. coli containing Moloney murine leukemia virus reverse transcriptase gene from clone of Moloney murine.

Unit definition

         Using Poly (A) as the template and oligo (dT) as the primer, the enzyme required to catalyze the incorporation of 1 nmol of dTTP within 10 minutes at 37°C is defined as one active unit (U).

Quality control

1. Absence of Endonuclease
   1. 1μg of Lambda DNA is incubated with 200 units of MMLV Reverse Transcriptase, RNase H Minus, 1×RT Reaction Buffer for 16 hour at 37°C. Following incubation, Lambda DNA is visualized as intact on an ethidium bromide-stained agarose gel to verify the absence of visible Endonuclease.
2. Absence of Nickase
   1. 1μg of Type I supercoiled pBR322 is incubated with 200 units of MMLV Reverse Transcriptase, RNase H Minus, 1×RT Reaction Buffer for 16 hour at 37°C. Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting.
3. Absence of Exonuclease
   1. 1μg of Lambda DNA / Hind III Markers is incubated with 200 units of MMLV Reverse Transcriptase, RNase H Minus, 1×RT Reaction Buffer for 16 hour at 37°C. Following incubation, Lambda DNA / Hind III Markers is separated by 1% agarose gel and stained with ethidium bromide. Markers remain as intact bands without smearing.
4. Absence of RNase
   1. 1ug of RNA is incubated with 200 units of MMLV Reverse Transcriptase, RNase H Minus, 1×RT Reaction Buffer for 4 hour at 37°C. Following incubation, the RNA is visualized as intact band on an ethidium bromide-stained agarose gel to verify the absence of visible RNase.
5. Function Assay
   1. First-Strand cDNA Synthesis 200 units of enzyme incubated with 2.5ug of 8.3kb RNA for 1 hours at 42°C must synthesize ≥65% cDNA. ≥45% of the cDNA must be >6kb when analyzed by agarose gel electrophoresis.
6. Physical Purity
   1. The purity is ≥95% as judged by SDS-polyacrylamide gel with Coomassie blue staining.