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Assay of gene transcription in vitro
1nterpretation of results and potential problems
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acid acrylamide activity affinity chromatography agarose gel ammonium analysis autoradiography buffer containing cDNA cells centrifuge cloning competitor DNA concentration denaturing described in Protocol digestion dilute DNA affinity DNA binding DNA fragment DNA polymerase DNA probe DNA-binding proteins DNase double-stranded EDTA EDTA pH 8.0 eluted Equipment and reagents ethidium bromide exonuclease expression filter footprinting fractions gel electrophoresis gel mobility shift gene glycerol hybridization Incubate labelled DNA lane ligation Method mg/ml MgCl2 microcentrifuge tube mixture mobility shift assay molecular molecules mutagenesis mutations NaCl nitrocellulose nuclear extract nuclease nuclease S1 nuclei nucleotide oligonucleotide pellet Phenol:chloroform:isoamyl alcohol plasmid plasmid DNA plates polyacrylamide gel preparation primer procedure promoter protein-DNA complexes Protocol 12 purified radiolabeled reaction remove restriction enzyme Resuspend RNase room temperature sample sequences single-stranded solution specific step supernatant TBE buffer template tissue transcription factor transfection transfer transgenic Tris-HCl pH vector vitro transcription wash yeast