StableScript Reverse Transcriptase

OEM by QIAGEN offers bulk manufacturing of StableScript Reverse Transcriptase in custom formulations.

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StableScript Reverse Transcriptase

Cat. No. / ID: P7720L

250 U (evaluation pack) of StableScript Reverse Transcriptase (5,000 U/ml) and 4X StableScript Reaction Buffer (1.5 ml)
Certain applications in which this product can be used may be covered by patents issued and applicable in the United States and abroad. Purchase of this product does not include a license to perform any patented application; therefore it is the sole responsibility of the users of the product to determine whether they may be required to engage a license agreement depending upon the particular application in which the product is used.
The StableScript Reverse Transcriptase is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Need this product modified? Contact us to get started with tailored OEM solutions.

Features

  • One-step RT-qPCR and long-range RT-PCR
  • Highly sensitive RNA detection
  • Improved thermostability, processivity and inhibitor resistance compared to first-generation M-MLV reverse transcriptase RNase H minus

Product Details

StableScript is a versatile reverse transcriptase designed for use in one-step RT-qPCR and long-range RT-PCR. The enzyme demonstrates high sensitivity for RNA detection, improved thermostability, processivity and inhibitor resistance over the first generation M-MLV reverse transcriptase RNase H minus.

Performance

StableScript Reverse Transcriptase is a versatile reverse transcriptase designed for use in one-step RT-qPCR and long-range RT-PCR. The enzyme demonstrates high sensitivity for RNA detection, improved thermostability, processivity and inhibitor resistance over first generation M-MLV reverse transcriptase RNase H minus.

Polymerase properties

·       Optimum extension temperature: 55°C

·       Transcription length: 12.3 kb

SDS purity n/a >99%
Single-stranded exonuclease 5,000 U <5.0% released 
Double-stranded exonuclease 5,000 U <1.0% released 
Double-stranded endonuclease 5,000 U No conversion 
E. coli DNA contamination 5,000 U <10 copies 
RNase contamination 5,000 U No detectable non-specific RNase 
RT-qPCR functionality n/a Amplification of test lot within 1 Cq of reference lot in a one-step RT-qPCR assay

Principle

Source of recombinant enzyme protein

The protein is produced by a recombinant E. coli strain carrying the engineered reverse transcriptase gene. This novel reverse transcriptase has no detectable RNase H activity and has increased thermostability.

Unit definition

One unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid insoluble material in 30 minutes at 55°C using poly r(A)/oligo (dT) as a substrate.

Procedure

Quality control analysis
Unit activity is measured using a two-fold serial dilution method. Dilutions of enzyme were made in 1X StableScript Diluent and added to 50 µl reactions containing 20 µg/ml poly r(A) RNA, oligo (dT) DNA, 1X StableScript Reaction Buffer, 3H-dTTP and 250 µM dTTP and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26).

The functionality of the RT-PCR assay is evaluated by amplification of three mRNA transcripts in a one-step RT-qPCR assay. The amplification threshold (Cq) of the test lot is compared to a reference lot.

Protein concentration (OD280) is determined by OD280 absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein-of-interest band in the diluted sample.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for four hours at 37°C.

Double-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl enzyme solution incubated for four hours at 37°C.

Double-stranded endonuclease is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl enzyme solution that is incubated for four hours at 37°C.

E. coli 16S rDNA contamination is evaluated using 5 µl replicate samples of enzyme solution denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Non-specific RNAse contamination is assessed using the RNAse Alert 1-Kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.

The enzyme is supplied in 20 mM K3P04 (pH 7.0), 1 mM EDTA, 1 mM DTT, stabilizer and 50% glycerol

 

Applications

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.
* RT-qPCR
* cDNA synthesis
* Long-range RT-PCR
* Next-generation sequencing