Cat.-no	Description	Amount	Price €	Shop
105-100	MMLV Reverse Transcriptase	10.000 units	80.00	add
105-250	MMLV Reverse Transcriptase	5x10.000 units	260.00	add

MMLV Reverse Transcriptase: Order/request by E-mail:
MMLV Reverse Transcriptase: Datasheet
MMLV Reverse Transcriptase: Deutsche Beschreibung

Applications:
- RT PCR
- Synthesis of cDNA
- mRNA 5’-end Mapping by Primer Extension Analysis
- End-labeling of DNA
- Dideoxynucleotide Sequencing

Description: 
MMLV Reverse Transcriptase, encoded by Moloney Murine Leukemia Virus (MMLV RT) is an RNA-dependent DNA polymerase that synthesizes the cDNA first strand from a single-stranded RNA template to which a primer has been hybridized. MMLV RT will also extend primers hybridized to single-stranded DNA. Second strand cDNA synthesis can be achieved from some mRNA templates without an additional DNA polymerase.  

Concentration: 200 u/µl

Storage Buffer:
200 mM potassium phosphate (pH 7.2), 0.2% Triton X-100, 2 mM DTT and 50% glycerol

Reaction Buffer 5X:
250 mM Tris-HC1(pH8.3), 375 mM KCl, 15 mM MgCl₂  and 50 mM DTT

Unit definition:
One unit of the enzyme incorporates 1 nmol dTTP into acid-precipitable material in 10 minutes at 37°C, using poly(A) oligo dT as a template primer.

Quality control:
Endonuclease Activity:  1 µg of Type 1 supercoiled plamid DNA is incubated with 500 units of enzyme in 1X reaction buffer for one hour at 37°C. The supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify absence of nicking or cutting. 
Nuclease Activity:  50 ng of radiolabeled DNA or RNA is incubated with 200 units of enzyme in 1X reaction buffer for one hour at 37°C, resulting in <1% release for both DNase and RNase.
Purity:  >90% as judged by SDS-polyacrylamide gels with blue staining. MMLV RT is free of detectable RNase, and DNase (exo- and endonuclease) activities.

Usage:  
Standard Protocol:
We recommend to prepare 2 Mixes

Mix I

Component	Amount/conc.
a. Total RNA  or  b. PolyA RNA  c. Strand-specific primer   or  d. oligo dT / random primer      for each µg of RNA	1-5 µg  50-500 ng  10 pM    250-500 ng
sterile Water	up to 8 µl
Incubation	Temperature
10 min	70 °C
10 - 15 min (for c. specific primers)  or  5 min (for d. oligo dT / random primer )	room temperature  place on ice

Mix II

Component	Amount/conc.
5X reaction buffer	4 µl
dNTP mix (10 mM of each = 40 mM)	1 µl
optional: RNAsin	20-40 units
MMLV Reverase (200 u/µl)	200 units
sterile water	up to 20 µl
combine Mix I and Mix II and gently vortex

Step	Temperature
30 - 115 min 1.)	37 - 55°C 2.)
10 min (Inactivation of enzyme)	65-70°C

^1.) 30 min for cDNA with 500 bp; 115 min for 1,5 kb
^2.) depends on the RNA: Higher temperatures (up to 55 °C) for higher structured RNA; Try to adjust the pH to 8.8

Transportation: on blue ice

Storage: at -20°C for 24 months

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