By Robert T. Sauer (Eds.)
Equipment popular within the examine of DNA binding proteins are provided during this quantity. those comprise purification and protein characterization, assays of protein-DNA binding and protein-induced bending, and biochemical and genetic equipment for probing the constitution, power, and specificity of protein-DNA interactions
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Mimori, J. A. Hardin, and J. A. Steitz, J. Biol. Chem. 261, 2774 (1986). 16 PURIFICATION AND CHARACTERIZATION  the previous section for the design of the oligodeoxynucleotides. The oligodeoxynucleotides should be purified by electrophoresis in a polyacrylamide-urea gel. 6, containing 100 mM MgC12,50 mM dithiothreitol (DTT), 1 mM spermidine, and 1 mM EDTA. Store at 4°. 6, containing 100 mM MgCl2, 150 mM DTT, and l0 mM spermidine. Store at 4°. ) 5'-Phosphorylation of Oligodeoxynucleotides 1. Combine 65/xl DNA (250/xg of each oligodeoxynucleotide in TE) with 10/zl of 10x T4 polynucleotide kinase buffer.
Dissolve the cyanogen bromide in 2 ml of N,Ndimethylformamide. It will instantly dissolve. Add the cyanogen bromide solution dropwise over 1 min to the stirring slurry of Sepharose in the fume hood. 4. Immediately add NaOH as follows. 8 ml. 5. Immediately add 100 ml of ice-cold water to the beaker and pour the mixture into a 60-ml coarse-sintered glass funnel. At this point, it is very important that the resin is not suction filtered into a dry cake. If the resin is accidentally dried during suction filtration, it will be necessary to start the CNBr activation procedure again from the beginning.