PCR‐generated DNA was translated into protein using the TnT® T7 Quick Kit (Promega). The TnT® quick master mix was thawed and other components were thawed at room temperature and thereafter stored on ice. The final volume of each reaction was 50 μl and included 40 μl of TnT® quick master mix, 1 μl of 1 mm methionine, 0.5 μg of PCR‐generated DNA template, 1 μl of T7 TnT® PCR enhancer and nuclease‐free water. The reaction was incubated at 30 °C for 90 min and stored at −80 °C.
Tnt t7 quick kit
Manufactured by Promega
Sourced in United States
The TnT® T7 Quick Coupled Transcription/Translation System is a cell-free protein expression kit. It allows for the rapid in vitro synthesis of proteins from DNA templates under the control of the T7 RNA polymerase promoter.
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Lab products found in correlation
2 protocols using tnt t7 quick kit
1
In Vitro Protein Translation from PCR DNA
2
In Vitro Binding Assay of SETD1A
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For in vitro binding assay, His-SETD1A (SET domain) was purified using Ni-NTA agarose beads. Glutathione S-transferase (GST)-fused SOX2 protein (a.a. 1-317, 1-204, 1-120, and 1-40) and SET domains of SETD1A (Win-SET, a.a. 1450-1707 and SET, a.a. 1538-1707) were expressed in BL21-DE3 and purified using glutathione-agarose beads. Full-length SETD1A was synthesized using the TNT T7 Quick kit (Promega, Madison, WI, USA). For the co-immunoprecipitation assay, cell extracts were prepared using a lysis buffer (20 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1% NP-40, and 2 mM EDTA) 41 (link). The SETD1A antibody was immobilized on Protein A/G agarose beads (Santa Cruz Biotechnology, Dallas, TX, USA) and incubated overnight with cell extracts at 4 °C. The beads were washed with lysis buffer and analyzed via western blotting.
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