By H. P. Saluz, J. P. Jost (auth.)
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Additional info for A laboratory guide for in vivo studies of DNA methylation and protein/DNA interactions
After quenching of the chemical re action the DNA is isolatcd and further processed in vitro. For all these procedures it is essential to optimize the incubation conditions for each cell system, Le. , 1988; Saluz and Jost, 1989). An example of in vivo DMS footprinting is given in Fig. 2. u 12345G ---------~--------- "'CTAG Fig. 2: Example of an in vivo DMS footprint: The left panel shows the four control reactions (G, A+G, T +C, C) carried out on cloned plasmid DNA containing the target sequence.
56). JII GewJlnic FO()lprinling D Dimethylsulfate (DMS) Treatment of Cells in Suspension or in Monolayer Cultures. When required, treat the cells in suspension with various amounts ofDMS or UV light. Use a large excess of the appropriate hormone (up to 10-7 M final) ifnecessary. Monolayer cell cultures can directly be used for footprinting experiments without disturbing the cell to cell contacts which are important in certain cases. Attention: DMS is very toxic and should only be handled in a well-ventilated fume hood (see page.
Incubate exactly 5 rnin at 20"C. > Add 10 ml ofice-cold DMS stop buffer and vortex gently. > Spin cells 5 min at 1000 x g (2500 rprn for HB-4 Sorvall rotor or equivalent) at 4"C. > Wash cells by resuspending in 10 ml ofnMS stop buffer. First add 5 ml, vortex briefly and add the other 5 ml aliquot. > Centrifuge as above. 5 rnl of cold nucIei buffer. 5 ml of cold nucIei buffer containing 1 % NP-40 and vortex weIl. > Keep suspension for 5 min on ice. > Centrifuge crude nuclei for 5 min at 4000 rpm (HB-4 Sorvall rotor or equivalent) at 4°C.