Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...

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tua@ats aftJ tttdi'ns
15oO rpm io collect ths cells. Media Ms asPiratsd and penet was
washed with 10 ml of PBs (PbosPhaie buifered saline) cells wer€
resuspended in 1 volume of digesion butler (1!-r mM NaCl' 10 mM
Tis/Hcl, pH B.o, 25 mM EDTA o 5% sos 200 rqlmL
proteinase K;
noler SDS and prot€ifase K wer€ added treshlv lo a stock-soldon
conlainng the rcmaining componenis) A lix€d volume ot digeston
blrller (3OO rli lhis volume will accomodats up lo 3x107 cells) was used
and fie suspension was incubated overnight ai 37'C The aqueous
solution conbining |he genomic DNA was eiracted with an equal
voLume ol phenoL chlorotom ad dvact€d onco wilh chLorotorm
isoamyl alcohol The DNA was precipjded by the addition ol 3tl
sodilm acetat€ (NaOAc) and 2 5 volumes oi ethanol Al lhis stage' the
qenomic DNA was kspl in elhanol and slored at -20'c lof long lerm
storage. For subsequenl use, |he precipnate would be collected bv
spinning lhe samples at 14,oOO rpm lor 15 minules at 4'c in a micro'
centituqe The pellst was washed with 70% elhanol an dried' and
.esusPended i. TE-builer.
App.oximately 20 /g ol genomic DNA lrom individual cell lines was
dillted lo 3oo ul in TE butler' The genomic DNAS wefe digested with
Hind lll (NEB bufi€r 3) and the sampl€s were incubatsd at 37'C
ov€rnght with o.e unit per l,g ot DNA. Aller th€ rgskiction dlqestion'
rhe samples were trealed with DNas€ t r.ee RNase 00 mg/mD lor 30
minules. subsequen{v, the samples were subjected Io elnanol