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

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

By H. P. Saluz, J. P. Jost (auth.)

ISBN-10: 3034877242

ISBN-13: 9783034877244

ISBN-10: 3764323698

ISBN-13: 9783764323691

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Extra info for A laboratory guide for in vivo studies of DNA methylation and protein/DNA interactions

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1 mM CoCb (the buffer is sterilized by filtration, kept frozen in aliquots and the enzymes are freshly dissolved before use). > Water bath shaker (3rC). 5 M EDTA, pH 8. 15 M NaCl, 20 mM Hepes (pB 8), 5 mM EDTA. > Sorvall centrifuge with HB-4 rotor or any equivalent. 01 M KHC03. > Dulbecco's culture medium (can be purchased). 52 III Genomic Footprinting Step-by-Step Procedure Preparation of Cell Suspensions from Adult Chicken Liver, Oviduct or Kidneys > LIVERS: perfuse Iivers with cold saline solution until they are bleached completely.

At maximum speed. 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.

8-1 % agarose gels (using appropriate size standards). Staining with ethidium bromide will show whether the DNA fragments are large enough without degradation. If the DNA is partially or completely degraded (Le. on agarose gel presenting a smear) do not use it for genomic sequencing. Ifthe D NAis contaminated with RN A, treat it again with ribonuclease. Opalescence ofthe DNA solution could be due to contamination with glycogen. In this ca se precipitate the DNA with isopropanol and wash the DNA pellet in :5 M sodium acetate, pli 5.

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


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