T7 P&L RNA Polymerase HC

Bacteriophage T7 RNA Polymerase is a DNA-dependent RNA polymerase (99 kDa) that catalyzes in vitro RNA synthesis from a DNA sequence containing a T7 phage promoter. The highly concentrated version and modified T7 RNA Polymerase HC is ideally suited for set-up and optimization of high yield in vitro transcription reactions. Proline 266 has been replaced by leucine (P266L) which has been associated with decreased abortive transcription[1].increased 5′ homogeneity of transcripts synthesized from A-initiating phi2.5 promoter[2]increased 5′ incorporation efficiency of GTP analogs[3] The provided T7 IVT Set-up Buffer, MgCl2 and DTT solutions enable testing of different reaction conditions such as nucleotide/ MgCl2 concentrations. Recommendation for assay set-up with minimal optimization: HighYield T7 P&L RNA Synthesis Kit (#RNT-201). Activity: 1 µl T7 P&L RNA Polymerase HC generates >/= 30 µg RNA in 10 min at 37°. This is comparable to 1000 U of NEB’s T7 RNA Polymerase (High Concentration), #M0460T (activity assay conditions: 1x T7 IVT Set-up Buffer supplemented with 10 mM DTT, 12 mM MgCl2, 3 mM NTPs, 1 µl T7 RNA Polymerase HC and 1 µg DNA template (1.4 kpb RNA transcript) in 20 µl. RNA yield was determined with a fluorescence microplate assay.)