TnT® Quick Coupled Transcription/Translation System Technical Manual
Literature # TM045
The TnT® Quick Coupled Transcription/Translation Systems are convenient single-tube, coupled transcription/translation reactions for eukaryotic in vitro translation. The original TnT® Coupled Reticulocyte Lysate Systems simplified the process and reduced the time required to obtain in vitro translation results compared with standard rabbit reticulocyte lysate systems. Standard rabbit reticulocyte systems commonly use RNA synthesized in vitro from SP6, T3 or T7 RNA polymerase. The TnT® Quick Coupled Transcription/Translation System further simplifies the process by combining the RNA polymerase, nucleotides, salts and Recombinant RNasin® Ribonuclease Inhibitor with the reticulocyte lysate to form a single TnT® Quick Master Mix. For most gene constructs, the TnT® Quick reaction produces significantly more protein (two- to sixfold) in a 60- to 90-minute reaction than a standard in vitro rabbit reticulocyte lysate reaction using RNA templates. The TnT® Quick Coupled Transcription/Translation System is available in two configurations for transcription and translation of genes cloned downstream from either the T7 or SP6 RNA polymerase promoters. Included with the TnT® Quick System is a luciferase-encoding control plasmid and Luciferase Assay Reagent, which can be used in a non-radioactive assay for rapid (<30 seconds) detection of functionally active luciferase protein. Starting with either circular plasmid DNA or PCR-generated DNA, in vitro transcription/translation results may be obtained easily in 5–6 hours.
Summary of Change
The following changes were made to the 8/22 revision of this document:
1. Section 1 was updated.
2. Section 3 was modified to provide cloning hints and additional details on plasmid use.
3. Sections 4.A–C and 5 were combined into Section 4.A. Subsequent sections were renumbered.
4. Section 7.B, Determining Percent Incorporation of Radioactive Label, is now Section 9.C.
5. The cover image and font were updated.