By H. P. Saluz, J. P. Jost (auth.)
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Extra resources for A laboratory guide for in vivo studies of DNA methylation and protein/DNA interactions
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. 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.
T: Flow diagram of DMS footprinting III Genomic Pootprinting 47 tissue did not impair its function. This can be tested by a combination of different techniques as indicated in the flow dia gram of Fig. g. in situ hybridization with suitable DNA or RNA probes, run ofT experiments with nuclei isolated from such cell preparations or in situ detection of specific antigens with antibodies. For all in vivo footprinting procedures published so far, cell suspensions were treated with either dimethylsulfate (DMS) , formaldehyde or UV -light.
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.