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You are here : RT-PCR - Reverse Transcription ╗ MMLV Reverse Transcriptase

MMLV Reverse Transcriptase

MMLV-Reverse Transcriptase: The enzyme that synthesizes a complementary DNA strand initiating from a primer using either RNA (cDNA synthesis) or single stranded DNA as a template

 
 

 Cat.-Nr:  Description  amount  Price € Shop
 105-100  MMLV Reverse Transkriptase     10.000 units   59.00   add 
 105-250  MMLV Reverse Transkriptase     50.000 units  229.00  add

MMLV Reverse Transkriptase: Request/Anfrage per E-mail

MMLV Reverse Transcriptase: Datasheet

MMLV Reverse Transkriptase: 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 plasmid 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 radio labelled 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


  

>>> Service pages in English <<< >>> Serviceseiten auf Deutsch <<<
Copyrightę GeneOn 2007-14 print page: MMLV Reverse Transcriptase
 


 
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Reverase Transcription

MMLV Reverse Transcriptase
MMLV Reverse Transcriptase, encoded by Moloney Murine Leukemia Virus (MMLV RT) is an RNA-dependent DNA polymerase that synthesizes the complementary cDNA first strand from a single-stranded RNA template to which a primer has been hybridized
AMV Reverse Transcriptase
AMV Reverse Transcriptase, encoded by Avian Myeloblastosis Virus reverse transcriptase and expressed in E.coli. is an RNA-dependent DNA polymerase that synthesizes the complementary cDNA first strand from a single-stranded RNA template
Ribunuclease Inhibitor
RNase Inhibitor is a recombinant human placental protein which specifically inhibits ribonucleases (RNases) A, B and C
Tth DNA Polymerase
Tth DNA Polymerase Maximo is used to reverse-transcribe RNA efficiently in the presence of manganese. PCR can be performed in the same tube using the intrinsic DNA polymerase activity simply by chelation of manganese cation and the addition of magnesium.


 
 
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