A laboratory guide for in vivo studies of DNA methylation by H. P. Saluz, J. P. Jost (auth.)

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

Example text

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. 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.

Put the suspension inlo an Erlenmeyer flask and incubate at 3TC for 5-10 min in a waterbath shaker. > Filter the digest with a nylon grid (1-mm-wide mesh) assembled into a funnel so that the filtration can be done into a glass beaker. > Rinse the filter with cold llank's solution. > Centrifuge cells at O'C at 300 x g (1500 rpm using a Sorvall HB-4 rotor or equivalenl) for 3 min. > Wash cells 3 times with ice cold Hank's solution as above. > If the pellet contains too many red blood cells, resuspend it in 10 ml ofred celllysis buffer (room temperature).

5 ml) add 30 fll of a 10x restrietion buffer (use the same buffer recommended by the manufacturer but without bovine serum albumin; the sterile filtered 10x buffer is stored at -20°C). Add the 15 flg of genomic DNA, mix gently by tapping the tube. Add sterile water up to a final volume of 300 fll per tube. Mix gently by tapping the tube ami add 45 units ofthe chosen restriction enzyme. Should restricLion enzymes with 4 bp recognition sequences be used, 150 units will be needed. Mix again as described above and give a shorl spin of a few seconds in a microfuge.

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