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RobusT™ II RT-PCR Kit

(M-MuLV RT RNase H-/DyNAzyme EXT) - A choice for low abundance RNA

 

  Description

RobusT™ II Kit is a high fidelity one-tube system with excellent performance and sensitivity. First-strand synthesis is catalysed by Moloney murine leukemia virus (M-MuLV) reverse transcriptase, RNase H-. Subsequent second strand PCR is performed by DyNAzyme™ EXT DNA Polymerase, a unique blend of thermostable DNA polymerase and proofreading enzyme.

M-MuLV RT RNase H- is purified from a recombinant source and due to the absence of RNase H activity it yields much larger quantities of full-length cDNA transcripts than wild type M-MuLV RT, which possesses substantial RNase H activity. RobusT™ II RT-PCR Kit offers an optimal enzyme combination for synthesis of long cDNAs from a variety of RNA sources and target sizes.

 

Applications

bulletDetection of gene expression
bulletQuantification of gene expression
bulletGeneration of cDNA products with high fidelity for cloning and sequencing
bulletClinical diagnostics: detection and quantification of infectious micro-organisms, cancer cells and genetic disorders.

 

Advantages

bulletHigh sensitivity of M-MuLV reverse transcriptase, RNase H-
bulletCapable of amplifying long targets, up to 6.0 kb
bulletOne-tube system: rapid, sensitive and reproducible analysis of RNA with minimal risk of sample contamination
bulletMinimal hands-on time required
bulletAmplifies products from a wide variety of total RNA or mRNA sources
bulletSaves time by eliminating the need to construct and screen cDNA libraries

 

Components

The kit contains sufficient materials for 100 (F-590L) or 20 reactions (F-590S) including control reactions.

RobusT™ II RT-PCR Kit

Component

Concentration

F-590S

F-590L

       

10 x RobusT™ Reaction Buffer

 

1.5 ml

1.5 ml

MgCl2 solution

50 mM

1.5 ml

1.5 ml

dNTP mix

10 mM each

20 µl

100 µl

M-MuLV Reverse Transcriptase, RNase H-

5 U/ µl

40 µl

200 µl

DyNAzyme™ EXT DNA Polymerase

1 U/ µl

40 µl

200 µl

Upstream Control primer

0 pmol/µl

20 µl

20 µl

Downstream Control primer

10 pmol/µl

20 µl

20 µl

Control RNA with carrier

10 ng MS2 RNA/µl

20 µl

20 µl

 

30 µg E. coli ribosomal RNA/ml

   

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Storage stability

Store all kit components at -20 °C. For long term storage, the control RNA may be stored at -20 °C. The RobusT™ II RT-PCR System is stable for one year from the date of packaging when stored and handled properly.

ORDERING INFORMATION

RobusT™ II RT-PCR Kit

Reactions

 
       

F-590S

 

20 reactions

F-590L

 

100 reactions

   
Individual components also available separately  

M-MuLV Reverse Transcriptase, RNase H-

Size

Concentration

F-572S

 

10,000 U

200 U/µl

F-572L

 

50,000 U

200 U/µl

DyNAzyme™ EXT DNA Polymerase

   

with Optimized EXT Buffer and Buffer Pack for Mg2+ Optimization

Size

Concentration

F-505S

 

200 U

1U/µl

F-505L

 

1,000 U

1U/µl

 

Figures

Broader working range with RobusT™ II RT-PCR system

     

M

DNA/Hind III + řX174/Hae III Digest

1

556 bp

human ADP Ribosylation Factor 1 (10 ng human skeletal muscle total RNA)

2

1.0 kb

dystrophin (100 mg human skeletal muscle total RNA)

3

2.2 kb

dystrophin (100 mg human skeletal muscle total RNA)

4

4.1 kb

dystrophin (100 mg human skeletal muscle total RNA)

5

6.0 kb

dystrophin (100 mg human skeletal muscle total RNA)

M

 

DNA/Hind III + řX174/Hae III Digest

Higher sensitivity for low abundance RNA

 

RNA: Human skeletal muscle total RNA

 

Target: Human ADP Ribosylation Factor 1

 

Marker: DNA/Hind III + řX174/Hae III Digest

 

Each successive lane represents decreasing amounts of total RNA from human skeletal muscle ranging from 100 nanograms to 1 picogram.

   
   

 


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