Tnt t7 quick for pcr dna

DNA-binding domains (DBDs) of cichlid proteins (NR2C2 and RXRB) were predicted based on alignment and conservation to annotated human and mouse orthologs. M. zebra and N. brichardi individuals were sacrificed according to schedule 1 killing using overdose of MS-222 (tricaine) at The University of Hull, UK and University of Basel, Switzerland. Tissues were stored in RNA later using a 1:5 ratio. RNA was extracted from brain, liver, and testis tissues of adult M. zebra and N. brichardi using the RNeasy Plus Mini Kit (Qiagen), achieving RNA integrity (RIN) in the range of 8–10 (Agilent Bioanalyzer Total RNA Pico Assay). First-strand cDNA synthesis of DBD-specific regions was carried out using RevertAid H Minus Reverse Transcriptase (Thermo Scientific) and DBDs amplified (2-step RT-PCR) using Platinum Taq DNA Polymerase (Invitrogen) and the primers listed in Additional file 1: Table S1. Resulting cDNA was concentrated using Minelute PCR purification (Qiagen) and 700 ng used for in vitro transcription/translation using TnT T7 Quick for PCR DNA (Promega) and the Fluorotect GreenLys tRNA (Promega) labelling system. DBD expression was resolved by SDS-PAGE and detection using the fluorescein filter in the ChemiDoc Touch (Bio-Rad) system.

GST-conjugated mA3 protein was prepared with the wheat germ cell-free protein synthesis system by following the manufacturer’s protocol (CellFree Sciences). Viral protease and its mutants were generated with an in vitro transcription/translation system using TNT T7 Quick for PCR DNA (Promega). The DNA templates to be translated to ²⁰PR²⁰ or ²⁰G/C²⁰ protein in this system were prepared by PCR using p57(2LTR)Sp72(CAG) or p57(2LTR)Sp72(G2509C)(CAG) as the template and the following primers: 5’-GGATCCTAATACGACTCACTATAGGGAACAGCTGGGATGAGAGATTGCCCCAAGAAGCC-3’ (F2) and 5’-CTATAGAGGCACATCTGGCCCTTTTGAGG-3’. To make the DNA template for ²⁰PR105, p57(2LTR)Sp72(G2509C)(CAG) and the primers F2 and 5’-CTAAATTTGGGCTTTTAGTTTAGTCAGCAA-3’ were used. For ²⁰PR85 or ²⁰PR65, p57(2LTR)Sp72(CAG) and each primer set F2 and 5’-CTATACATGGAGGAAAGAGTGGGTGACCTT-3’ (for ²⁰PR85) or F2 and 5’-CTACCAGCGATACCGCTTTCCTCCAGTAGC-3’ (for ²⁰PR65) were used, respectively. Sixty μg of GST-conjugated mA3 bound on Glutathione Sepharose resin (GE Healthcare) was mixed with the in vitro transcribed/translated viral protease for 3 hours at 4°C with gentle agitation. The resin was washed with PBS containing 0.05% Tween-20 and protein complexes on the resin were dissolved in Laemmli SDS sample buffer. The resultant pulled-down proteins were analyzed by immunoblotting.

TNT T7 Quick for PCR DNA was from Promega (Madison, WI, USA). Dog pancreas ER rough microsomes were from tRNA Probes (College Station, TX, USA). EasyTag EXPRESS³⁵S Protein Labeling Mix, [³⁵S]-L-methionine and [³⁵S]-L-cysteine, for in vitro labelling was purchased from Perkin Elmer (Waltham, MA, USA). Restriction enzymes were from New England Biolabs (Massachusetts, USA) and endoglycosidase H was from Roche Molecular Biochemicals (Basel, Switzerland). PCR and plasmid purification kits were from Thermo Fisher Scientific (Ulm, Germany). All oligonucleotides were purchased from Macrogen (Seoul, South Korea).