APOBEC1-YTH-HA and APOBEC1-YTHmut-HA proteins were in vitro transcribed/translated using the Promega TNT T7 Quick Coupled In Vitro Transcription/Translation kit. Briefly, 1 μg of plasmid DNA was used in a 50 μl reaction and incubated for one hour at 30°C. 5 μl of each reaction was then mixed with 30 ng of a 1500 nt-long RNA with a single internal A or m6A site, 0.5 μl RNasin (Promega), in 1X deaminase buffer (10mM Tris-HCl pH7.5, 50mM KCl, 0.1uM ZnCl2). Reactions were incubated for 4 h at 37°C. RNA was isolated with the Qiagen RNeasy Plus Mini kit and treated with DNase I (New England Biolabs) for 15 min at 37°C. For assays using cellular RNA, in vitro deamination was carried out for 6 h at 37°C using 50 ng of total RNA from HEK293T cells. Sequencing libraries were then prepared using the NebNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).
Tnt t7 quick coupled in vitro transcription translation kit
Manufactured by Promega
The TNT T7 Quick Coupled In Vitro Transcription/Translation kit is a reagent system that enables the rapid production of proteins from DNA templates in a single-tube reaction. It couples the transcription and translation processes to efficiently synthesize proteins in a cell-free environment.
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3 protocols using tnt t7 quick coupled in vitro transcription translation kit
1
In vitro Deamination Assay for APOBEC1
2
In vitro Deamination Assay for APOBEC1
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APOBEC1-YTH-HA and APOBEC1-YTHmut-HA proteins were in vitro transcribed/translated using the Promega TNT T7 Quick Coupled In Vitro Transcription/Translation kit. Briefly, 1 μg of plasmid DNA was used in a 50 μl reaction and incubated for one hour at 30°C. 5 μl of each reaction was then mixed with 30 ng of a 1500 nt-long RNA with a single internal A or m6A site, 0.5 μl RNasin (Promega), in 1X deaminase buffer (10mM Tris-HCl pH7.5, 50mM KCl, 0.1uM ZnCl2). Reactions were incubated for 4 h at 37°C. RNA was isolated with the Qiagen RNeasy Plus Mini kit and treated with DNase I (New England Biolabs) for 15 min at 37°C. For assays using cellular RNA, in vitro deamination was carried out for 6 h at 37°C using 50 ng of total RNA from HEK293T cells. Sequencing libraries were then prepared using the NebNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).
3
Binding Assay of TRβ1 Mutants to p85α-CSH2
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Binding of TRβ1 or C-terminal mutants (PV, Mkar, Mdbs, and AM) to GST–CSH2 of p85α was carried out as described in [25 (link)] with modifications. In vitro translated TRβ1 and C-terminal mutants (PV, Mkar, Mdbs, and AM) were synthesized by using a TNTT7 quick coupled in vitro transcription/translation kit (Promega). E.coli expressed GST or GST fused p85α-CSH2 protein (~5 μg) was used in each binding reaction. Their concentration was determined by coomassie blue-stained band intensities using bovine serum albumin standards after migration in SDS-PAGE gel. For relative binding affinity studies, identical amounts of in vitro-translated TRβ1 and C-terminal mutants (PV, Mkar, Mdbs, and AM) were used. To that purpose, the bound proteins were analyzed by Western blotting using anti-TRβ1 antibody, and the band intensities of TRβ1 or C-terminal mutants (PV, Mkar, Mdbs and AM) were quantified with NIH IMAGE software (Image J 1.47v; Wayne Rashband, NIH).
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