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
The re action scheme is described in Fig. 2b. When used to investigate areas of proteinlDNA interaction, the technique is referred to as genomic footprinting with Taq polymerase, as described in part III of this book. :::::::::::::::: 3' 3' Fig. 2b: Reaction scheme: A DNA strand with 2 unmethylated and 1 methylated cytosine is shown. Upon subjection to a C-specific sequencing reaction, only the two unmethylated sites can be cleaved, resulting in the possible reaction products shown. No cleavage occurs at the methylated cytosine.
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). n DNA Isolation 37 > 38 Ifthe DNA is still contaminated with RNA, LreaL iL wiLh pancreaLic ribonuclease as outlined for ON A Preparation from Frozen Animal Tissues and Cell CulLures. R. Johnston (1988). 0/20 mM > Zymolase 60 000 (Miles Laboratories) > 30°C shaker > 10% SDS/Water > Proteinase K (freshly prepared in TEN bufTer) > Phenol/chloroform (1:1) > Chloroform/n-amyl alcohol (24:1) > Absolute ethanol > RNase A > Sterile glass rods 40 II DNA Isolation ~~I)TA Step-by-Step Procedure > Take 1 I 01' cells (laIe log phase, approximately 5x10 7 cells/ml ol'culture medium).
The right panel shows genomic footprints of the same target sequence obtained using different concentrations of dirn ethyl sulfate (DMS). Proteins bound to DNA protect these areas from modifications by DMS and subsequent chemical cleavage, leaving their "footprint" on the DNA. The resulting fragments can be detected by either ofthe genomic footprinting procedures. 01 M KIIC03 > Dulbecco's medium (can be purchased) > Trypan blue (vital stain; see Viable Cell Count in Appendix) III Genomic Footprinting 49 Step-by-Step Procedure > Take 18 day old chicken embryos and perfuse the livers in situ by injecting 5-10 ml 01' Hank's solution per chick into the heart.
A laboratory guide for in vivo studies of DNA methylation and protein/DNA interactions by H. P. Saluz, J. P. Jost (auth.)