~weigang/Lecture-syllabus.html
GeneGene productAssay lacZβ-galactosidaseHistochemical test uidAβ-glucuronidaseHistochemical test luxLuciferaseBioluminescence GFPGreen fluorescent protein Fluorescence Reporter Gene Assay
PITX2-LACZ Staining of mouse embryo day 11 PITX2: bicoid-related transcription factor
Luciferase gene Luciferase Activity (Promega)
Supplemental Figure S1. Schematic representation of the procedure for in vitro transcription and translation. The PCR products used as DNA templates for in vitro transcription, malTWT and malTP7, were obtained with an upstream primer containing the T7 promoter sequence (pT7) and a downstream primer including the codons for 8 histidine amino acid residues (8x His) followed by a stop codon (STOP) using the genomic DNA of E. coli strains pop1951malTWT and pop1953malTP7 (Danot and Raibaud, 1994). The DNA templates were transcribed in vitro using the Ribomax Large Scale RNA production system (Promega) producing mRNA malTWT and mRNA malTP7. The transcripts were translated using E. coli S30 extract prepared from E. coli strain BSN29 (hns, stpA) in the presence of 35S- methionine, with or without addition of H-NS or StpA, for 1 hour at 30 。 C or 37 。 C. A similar procedure was used in the other tested cases (i.e. dpiA, lrhA, znuA, yhbW. ynfF) and the translation products were truncated, C-terminally His-tagged, short peptides like those from malT. The radiolabeled translation products were resolved on 12.5% SDS-PAGE, visualized by autoradiography, and quantified with a ChemiDoc XRS (BioRad) using Quantity One software. The measured values are within the linear range of detection by ChemiDoc XRS.