Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...
Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...
Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...
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E2 F. cen crct. Connat al <strong>H4</strong> c*e Tansctipnon<br />
NaH2PO.6nd 315 ml ol 1M NatlPo.l' !o !g/ml denaturcd slmon<br />
sperm oNA and 500,6 lormald6hy@. Th8 m6mbran6s wers incubated<br />
wirholr radiolabeLed DNA in pr€.hybridizalion sollllon tor o.e rrour 3l<br />
65'c. This solullon was renov€d lrom lho cylind€r and .eplac€d by 3_4<br />
ml hybridi2alion solulion [5 x sSC, 5 x Denhardi's soiulion,002 M<br />
phosphare bufer. pH 6.71, gl)% to,maldehyd€ Badio'lab€led DNA<br />
Prcbe (2x1@ cpm/ml) was boiled inth6 P€sence oi 100,rglmlsahon<br />
spefm oNAlor l0 minltes and add6d tothe hybridyzal on mlxtlre 'rhe<br />
membranes w€re hybridized lor 20. 24 hours at 42'C Subsequentlv,<br />
lhe memb.anes were rinsed twice with a wash_solution (6 x scc and<br />
0.1% sodium dodecfl sulphat€ lsDsl) at 65'c Each rinse was<br />
periormsd in a shaking wale. bath foi 30 minutes. The membrane was<br />
washecl o.ce wllh 2 x SSC 0.1% SDS al 65 C tor 15 minules.<br />
Membranes w6r€ died on a lilt€r Pap€r and subjed to €utoradiographv<br />
by ovemight exposure at -70'C using xiay f|m (Kod.k X_OMAI aR,<br />
Easrman Kodak). The onginal aga. plates on trt'ich lho coronres were<br />
qrown. wefe alig.ed lo lhe X-rsy li m for idenlilication and processing ol<br />
positve clones {see Fig.8)<br />
Small sc.le isolation orplasmid DNA<br />
Prasmid oNA lrom 2 ml cultures was prepared bv th€ boiling melhod as<br />
described previously (Hohes and Auigl€y, 1941). Singl€ colonies were<br />
lnoculared in 2 ml LB medum and incubated at 37'C ovornight, Alqlols ol<br />
each cullure (15 ml) were ransle(€d into 1.5 ml EpPendod Ubes and
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