This product is a bacteriophage T7 RNA polymerase derived from recombinant protein expression in Escherichia coli. It catalyzes the 5'→3' synthesis of RNA on double-stranded DNA from its T7 promoter sequence (5'-TAATACGACTCACTATAG*-3') and uses NTPs as substrates. The DNA with double-stranded linear blunt ends or 5'protruding ends can be used as templates for T7 RNA polymerase, so linearized plasmids and PCR products can be used as templates for in vitro synthesis of RNA. Note: G* is the first base of the RNA transcript. This product is produced in accordance with GMP regulations and provided in liquid form. Feature Synthesize long transcripts and short transcripts, RNA can be produced 100-200 μg with 1 μg of DNA template Higher yield and easy to operate Lower endotoxins Tested for the absence of endonucleases, exonucleases, RNases Application Radiolabeled RNA probe preparation RNA generation for studies of RNA structure, processing and catalysis Expression control via anti-sense RNA mRNA, sgRNA synthesis Specification Source Expressed in E. coli with a recombinant T7 RNA Polymerase gene Optimum Temperature 37℃ Storage Buffer 50 mM Tris-HCl, 1 mM EDTA, 10 mM DTT, 100 mM NaCl, 0.1% Triton X-100，50% (v/v) glycerin，pH7.9 at 25℃ Unit Definition The amount of enzyme required to incorporate 1 nmol of [3H] GMP into the acid-insoluble precipitate within 1 hour at 37°C and pH 8.0 is defined as 1 unit. Components Components No. Name 10624ES90 (5,000 U) 10624ES97 (50,000 U) 10624 T7 RNA Polymerase GMP-grade (50 U/μL) 100 μL 1 mL Shipping and Storage T7 RNA Polymerase GMP-grade products are shipped with dry ice and can be stored at -15℃ ~ -25℃ for one year. Figures Figure 1. Standard RNA was synthesized in vitro using T7 RNA synthesis kit. The reaction was incubated in a PCR instrument at 37℃ for 2h and then purified by magnetic beads (Cat#12602). The yield result was analyzed by NanoDrop spectrophotometer as shown in Figure 1. Figures 2. The transcription demonstration of different lengths RNA by T7 kit respectively in the electrophoretogram (Figures 2A), the capillary electrophoresis diagram (Figures 2B), and the chromatogram (Figures 2C) Figure 3. Synthesis of capped RNA in vitro. The reaction was incubated in a PCR instrument at 37℃ for 2h and then purified by magnetic beads (Cat#12602). The yield result was assayed by NanoDrop spectrophotometer as shown in Figure 3A. The integrity result was analyzed by capillary electrophoresis as shown in Figure 3B. FAQ Q: How is the fidelity of the T7 enzyme? Have any related tests been conducted? A: Our T7 enzyme test has measured the relative fidelity, which is similar to that of Thermo. The mutation rate is approximately four parts per million. Currently, the fidelity of the wild-type T7 is quite similar and can basically meet the needs of customers. There is no plan for developing a higher-fidelity T7 in the current project. Q: Was the yield measured using T7 polymerase after LiCl purification? Are there data for A260/280 and A260/230? A: The measurement was conducted after LiCl purification. We have recorded the relevant data. The ratio of A260/280 is approximately within the range of 2.0 - 2.1; using our carboxyl magnetic beads for purification also falls within this range. The specific data can be compiled and shared later. Q: When using T7 polymerase for transcription, is there any way to reduce the problem of excessive polyA tailing? A: This situation is an inherent characteristic of the T7 reaction system and cannot be completely avoided. According to relevant literature, the main approaches to controlling the self-extension of mRNA are as follows: Optimize the upstream sequence of the 3' end of the template; ② Control the output of IVT. Under high production levels, the probability of self-expansion will be higher. Documents: Safety Data Sheet MSDS_10624_HB220701 Manuals 10624_Manual_HB221111 Related blog： DNase I Applications
