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
2 g ofMacerozyme R 10 (Yakult Honsha Co Ltd) in 20 ml of Sorbitol butTer. Add 10 ml enzyme solution to each petri dish. > Incubate at 30"C for 4 h, without agitation. 2-mm mesh filter. Wash residue on the filter 3 times with Sorbitol butTer to release all protoplasts. In this and subsequent steps keep everything on ice. > Centrifuge protoplasts at 100xg (1000 rpm, OS3 rotor from Sorvall or equivalent) for 5 min. > Resuspend protoplasts in 20 ml sucrose butTer. Transfer to 30-ml Corex centrifuge tubes.
Transfer cell suspension into a 15 ml Corex centrifuge tube, pipet up and down a few times and leave the suspension for 5 min at room temperature. 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.
Of 3 M sodium aceLate, pli 5. > Mix and add 1 vol. 01" isopropanol. Mix well by II DN/1 Isolation 33 inversion and the DNA will precipitate immediately. 34 > Take out thc clump ofgenomic DNA with a sterile glass rod and put it into an Eppendorf tube. 2 M NaCI by centrifugation. > Remove the residual ethanol with a flow ofnitrogen but do not dry the pellet of DNA. 5 mM EDTA and let DNA dissolve overnight at4°C as outlined forthe preparation ofDNA from frozen tissue. Il DNA isolation E DNA Preparation from Plant Protoplasts (Barley Seedlings) This procedure has been described in the PhD thesis of Mikael BIom Sorensen at the Carlsberg laboratory, Department of Physiology, Copenhagen DK 2500 (with the permission of the director Prof.