Cat. No. / ID: P7720L
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.
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.
· Optimum extension temperature: 55°C
· Transcription length: 12.3 kb
|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|
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.
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.
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
This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.
* cDNA synthesis
* Long-range RT-PCR
* Next-generation sequencing