StableScript™ is a versatile reverse transcriptase designed for use in One-step RT-qPCR and Long Range RT-PCR. It 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: 12.3kB
Molecular weight: 115,035 Daltons
Source of Protein: 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 1nmol of dTTP into acid insoluble material in 30 minutes at 55°C using poly r(A)/oligo (dT) as a substrate.
Unit Activity is measured using a two-fold serial dilution method. Dilutions of enzyme were made in 1X StableScript Diluent and added to 50µL reactions containing 20µg/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 (Ct) 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 50µL reaction containing a radiolabeled single-stranded DNA substrate and 10µL of enzyme solution incubated for four hours at 37°C.
Double-Stranded Exonuclease is determined in a 50µl reaction containing a radiolabeled double-stranded DNA substrate and 10µL of enzyme solution incubated for four hours at 37°C.
Double-Stranded Endonuclease is determined in a 50µL reaction containing 0.5µg of plasmid DNA and 10µL of enzyme solution incubated for four hours at 37°C.
E.coli 16S rDNA Contamination is evaluated using 5µL 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.
Supplied in: 20mM KPO4, pH 7.0, 1mM EDTA, 1mM DTT, stabilizer, 50%glycerol
Supplied with: 4X StableScript Reaction Buffer
|Unit Size||250 U|
|4X StableScript Reaction Buffer||1 X 1.5 mL|
|Unit Size||250 units|
|SDS||Available on request|
|Storage Temperature||-25⁰C to -15⁰C|
|SS Exonuclease||5,000||<5.0% released|
|DS Exonuclease||5,000||<1.0% released|
|DS Endonuclease||5,000||No conversion|
|E.coli DNA Contamination||5,000||<10 copies|
|RNAse Contamination||5,000||No detectable non-specific RNase|
|Functional RT-PCR Assay||N/A||Amplification of test lot within 1Ct of reference lot in a one-step RT-qPCR assay|
* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.
|Product Description||Part Number|
|Phoenix Host Start Taq DNA Polymerase||P7590L|
Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.
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