This product is a bacteriophage T7 RNA polymerase derived from recombinant expression in E. coli. It is a T7 RNA polymerase obtained by mutation after modification and optimization of wild-type T7, which can significantly reduce the dsRNA content. Double-stranded DNA containing the T7 promoter sequence is used as a template and NTP is used as a substrate to synthesize RNA complementary to the reverse single-stranded DNA downstream of the promoter. Double-stranded linear blunt-end or 5' protruding end DNA can be used as a substrate template for T7 RNA polymerase, so linear plasmids and PCR products can be used as templates for in vitro RNA synthesis. Specifications Source Recombinant E. coli Optimum temperature 37℃ Activity 1000 U/μL Residual RNase Negative Storage buffer 50 mM Tris-HCl, 1 mM EDTA, 10 mM DTT, 100 mM NaCl, 0.1% Triton X-100, 50% (v/v) glycerol, pH7.9@25℃ Activity unit definition The amount of enzyme required to incorporate 1 nmol of [3H] GMP into the acid-insoluble precipitate within 1 hour at 37℃ and pH8.0 is defined as one activity unit. Components Components No. Name 10636ES50 (50 KU) 10636ES60 (500 KU) 10636ES72 (1 MU) 10636ES86 (10 MU) 10636 Hieff™ T7 RNA Polymerase (low dsRNA, 1000 U/μL) 50 μL 500 μL 1 mL 10 mL Figures 1. Customer feedback 1 Experimental Design: Parameter Condition Objective To compare application performance between WT T7 and Low-dsRNA T7 Reaction Volume 20 µL Template Length 2 kb Test Samples WT T7 & Low-dsRNA T7 RNA polymerase Reaction Temperature 27 °C Reaction Time 2 h Results: Table 1. Comparison of dsRNA Content and Yield Between WT T7 and Low-dsRNA T7 Enzyme Type dsRNA Content (%) Integrity (%) Reaction Scale WT T7 0.0030% 92.5 180 µg (20 µL) Low-dsRNA T7 0.0003% 93.0 180 µg (20 µL) Result Summary: Low-dsRNA T7 reduced dsRNA content by approximately 10-fold while maintaining comparable RNA yield and integrity relative to WT T7. 2. Customer feedback 2 Table 2. Performance Comparison Under Different GAG Final Concentrations Yield (mg/mL) GAG Final Concentration (mM) Yield (mg/mL) Integrity (%) Capping Efficiency (%) Transfection Efficiency (%) WT Yeasen supplier A* WT Yeasen supplier A* WT Yeasen supplier A* WT Yeasen supplier A* 10 12.14 11.376 12.103 88.1 92.5 92.3 96.7 100 100 44.60 33.63 30.76 5 11.82 10.716 11.337 82.2 91.1 91.5 98.3 100 100 42.30 32.78 32.37 2.5 11.31 10.716 11.066 82.9 91.7 91.8 98.8 97.3 97.8 39.78 31.68 32.59 1.5 11.98 — — 78.9 — — 97.7 — — — — — 1 11.75 — — 80.0 — — 90.5 — — — — — 0.5 11.65 — — 80.9 — — 70.1 — — — — — Result Summary: Effects of cap analog concentration on IVT performance. Reactions were carried out in a 20 µL system at 27 °C for 2 h using different T7 enzymes. RNA yield remained stable as cap analog input decreased, while integrity and capping efficiency showed a gradual decline. A cap analog concentration of ≥2.5 mM is recommended for optimal performance.
