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
Dialyze the DNA solution overnight against 10 I of glass-distilled water. > Add to the DNA solution 1/10 vol. of 3 M sodium acetate, plI 5. > Mix and add 1 vol. 01' isopropanol. Mix weil by inversion and the DNA will precipitate immediately. > Take out the c1ump 01' genomic DNA with a sterile glass rod and put it into an Eppendor1'tube. 2 M NaCI by centri1'ugation. > Rem(we the residual ethanol with a flow ofnitrogen but do not dry the pellet 01' DN A. 5 mM EDTA and letDNA dissolve overnight at 4°C as indicated previously in section B (page 28).
Transfer the aqueous phase with a sterile pipet (without toU(~hing the interphase) into a SW-40 or SW-27 polyallomer Beckman centrifuge tube. > Add 1/10 vol. 5 vols of ethanol. Mix weH by inversion amlleave it overnight at -20°C. > Centrifuge (25000-30000 rpm, 4°C, 1 h), decant the supernatant and dry the DNA sediment under vacuum. Dissolve the DNA in 200 111 of sterile distilled water. > Determine the OD:wo from an aliquot and store the DNA at - 70°C if it is not to be used immediately for the Maxam and Gilbcrt sequencing reactions.
Perform another 5-6 extractions as described above. > Do a final extraction with 5 ml chloroform (leave interphase), centrifuge as above and transfer aqueous phase in dialysis tubings. 5 mM EDT A in the cold changing the dialysis buffer once a day. > Determine the DNA concentration (OD260) and check the DNA quality on a 1 % agarose gel. Note: For monolayer cultures the DMS reaction, quenching and the rinsing of the cells occurs in situ in the tissue culture dishes. For DNA preparation use the direct lysis procedure as outlined in DNA Preparation from Frozen Tissues and Cells in Tissue Culture (p.