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
III Genomic Footprinting 57 E Short Protocol for DMS Treatment of Cells in Suspension or in Monolayer Cultures > Prepare 6 labeled 15-ml Corex centrifuge tubes in iee. > Add 1 ml 01' cell suspension (108 cells)/tube. > Add the appropriate amount or DMS while mixing slowly. > Incubate exactly 5 min at 20°C. > Add 10 ml 01' ice-cold DMS stop buffer. > Vortex gently. > Spin for 5 min at 1000 x g at 4°C. > Wash cells by resuspending in 10 ml of DMS stop buffer. > Centrifuge as above. 5 ml of cold nuclei buffer.
Add 2-5 ml Zymolyase 60000 (Miles Laboratories; 1 mg of Zymolyase per ml of TEN bufTer; always freshly prepared). > Incubate at 30"C in a shaker for 1 h (this time might be increased für strains more resistentto spheroplasLing. Spheroplasting can be tesled by mixing a drop 01' culture suspension in 1 ml of water. Clearing 01' the suspension upon addition of a drop of 1O'y" SDS/water indicates a high level of spheroplast conversion). > Centrifuge the spheroplasts at 3000xg for 5 min at 4"C. > Resuspend spheroplasts in S~~C bufTer and centrifuge für 5 min at3000xg and 4"C (spheroplasts may be stored at-20"C for several days).
Centrifuge crude nuclei for 5 min at 4000 rpm (HB-4 Sorvall rotor or equivalent) at 4°C. 5 rnl of nuclei buffer and transfer the suspension into a 15-ml sterile Falcon conical tube. > Add an equal volume of 2 x proteinase K buffer containing 600 ~g of proteinase K/rnl. > Seal the Ud ofthe tube with paral"ilm amI incubate 111 Genomic Footprinting the tubes in a horizontal position under water with reciprocal shaking at 37°C overnight. > Digest nuclear RNA by adding 50-100 Ilg ofpancreatic ribonuclease Aper ml and continue incubation as above for 1-2 h.
A laboratory guide for in vivo studies of DNA methylation and protein/DNA interactions by H. P. Saluz, J. P. Jost (auth.)