By H. P. Saluz, J. P. Jost (auth.)
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Extra info for A laboratory guide for in vivo studies of DNA methylation and protein/DNA interactions
Within this time red blood cells willlyze and cell cIumps will sediment to the bottom of the tube. > Carefully remove the cell suspension with a pipet, leaving behind the cell clumps at the bottom of the tube and put it into a 15 ml Corex centrifuge tube. > Centrifuge cells at 1000 x g for 6 mi n (O°C) as above. > Wash cells with cold bufTered saline amI centrifuge as above. > Resuspend cells in :) ml of Dulbecco's medium, adjust cell density to 101l cells/ml and use them immediately for footprinting (p.
15 M NaCI, 20 mM Hepes [plI 8], 5 mM EDTA). > Pour cell suspension into 30 ml Corex tubes and centrifuge at 1000 x g (2500 rpm for 11 13-4 rotor 01' equivalent), O°C for 5 min. > Decant supernatant and resuspend eells in buffered saline solution by carefully vortexing. > Filter cell suspension with a nylon grid funnel 1lI Genomic Footprinting 53 (mesh size: 1 mm) without teasing the surface of the filter. 54 > Centrifuge in the cold as indicated above. > If the pellet has too many red blo()() cells, resuspend it into 10 ml 01' red blood cell lysis medium at room temperature.
5 mM spermine (polyamines should be added just before use). 5% sodium dodecylsulfate. 1 'X, hydroxyquinoline). 32 II DNA Isolation Step-by-Step Procedure > Measure the approximate volume of the sperm suspension. > Wash sperm suspension 3 times with 100 mM EDTA, 100 mM Tris-HCI (pfl8) by centrifugation at 5000xg for 10 minutes at O"C (Sorvall centrifuge or equivalent). ,=DTA, 10 mM Tris-IICI (pI! 2 mM spermine. 5% SDS. > Incubate the solution at 50"C for 30 min. The sperm will swell and become permeable to the prolcinase K.
A laboratory guide for in vivo studies of DNA methylation and protein/DNA interactions by H. P. Saluz, J. P. Jost (auth.)