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TRANSCRIPT
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http://www.sciencegateway.org/resources/biologytext/dogma/trx.html
http://faculty.ksu.edu.sa/dr.afafshehata/Pages/Documents.aspx
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http://aprendendogenetica.blogspot.com/2010_04_01_archive.html
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http://www.uic.edu/classes/phys/phys461/phys450/ANJUM04/
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http://diverge.hunter.cuny.edu/~weigang/Lecture-syllabus.html
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A1167
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Gene Gene product Assay
lacZ β-galactosidase Histochemical test
uidA β-glucuronidase Histochemical test
lux Luciferase Bioluminescence
GFP Green fluorescent protein
Fluorescence
Reporter Gene Assay
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PITX2-LACZ Staining of mouse embryo day 11
PITX2: bicoid-related transcription factor
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Luciferase gene
Luciferase Activity (Promega)
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Supplemental Figure S1. Schematic representation of the procedure for in vitro transcriptionand translation.
The PCR products used as DNA templates for in vitro transcription, malTWT andmalTP7, were obtained with an upstream primer containing the T7 promoter sequence (pT7) and adownstream primer including the codons for 8 histidine amino acid residues (8x His) followed bya stop codon (STOP) using the genomic DNA of E. coli strains pop1951malTWT andpop1953malTP7 (Danot and Raibaud, 1994). The DNA templates were transcribed in vitro usingthe Ribomax Large Scale RNA production system (Promega) producing mRNA malTWT andmRNA malTP7. The transcripts were translated using E. coli S30 extract prepared from E. colistrain 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) usingQuantity One software. The measured values are within the linear range of detection by ChemiDocXRS.
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