T7 P&L RNA Polymerase HC

For general laboratory use.

Please centrifuge briefly before opening (volume ≤2 ml).

Description:
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 Proline 266 has been replaced by leucine (P266L) which has been associated with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.)

Shipping: shipped on gel packs

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months

Purity: ≥ 95 % (SDS-PAGE)

Form: liquid

Concentration: 2.0 – 2.2 μg/μl (A280, ɛ = 140 L mmol^-1 cm^-1 [1] in T7 RNA Polymerase HC Storage Buffer)

Content:
T7 P&L RNA Polymerase HC
1x 50 μl (2 μg/μl)
50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, 5 mM DTT, 0.1 % Triton-X-100, 50 % Glycerol (v/v), pH 8.0

T7 IVT Set-up Buffer
1x 200 μl (10x)
400 mM Tris-HCl (pH 8.0), 20 mM Spermidin

DTT
1x 100 μl (100 mM)

MgCl₂
1x 200 μl (200 mM)

Related products:

• HighYield T7 RNA Synthesis Kit, #RNT-101
• RNAse Inhibitor - recombinant, #PCR-392
• NTP bundle, #NU-1014

Selected References:
[1] Guillerez et al. (2005) A mutation in T7 RNA polymerase that facilitates promoter clearance. Natl. Acad. Sci. U.S.A102:5958.