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

kn f: c.n cyd. cdnot ot H4 6@0 rtanx'igion
bufier trom lhe column, While keeping lhs column in a straight position,
the bufier was allowed ro rrav€rse rhrough tho column by gravity ltow.
Th€ ljp ol the column was placed inside a coLtection rlbe and
centriluged a! 2500 rpm lor 2 mi.ut€s. The lir$ lube was discarded and
lhe collmn was paced of top of €nother cot€cUon tub€. Th€ probe
sorurion was added in th6 center of the s€phadex beads, and thg
sampre spun down lor 5 minltes at 2:@ rpm. The cotumn which
conlains the majority ol rhe free tab€twas discarded in rhe radioacrive
wasle bin (!h€ top cover and bortom tip clver were pur back on the
corumn ro prevent the spilling ot iadioacrive beads). The approximate
speciiic aclivity of rhe pfobe was delermined by anatyzing rhe yietd ot
radio-acrlvityr 1 rl was added in 4 mt of scintiltarion oprifluor and
counl€d in scinila on counter,
slandard procedure tor hybrtdtzalton
Hybn:dization was perlormed tn glass hybridization cytinders. prioi ro
use, lhe cylinders wer6 washed and siticonized wirh 5% siticonizing
solurion in chtoroiorm. rhe UV dosstinked memtanes (conlaining rhe
genomrc ONA from the c€tt lines) we.e removed tom the seated bag
roned up and placed ln lhe gass cytinder. To lhe cylinder 25 mt of
pfehybridization sotution was added (1 mt\,i ot EoIA, 0.25 t\4 NarHpq
pH 7.2 and 7% SOS) to coar rhe membrane, and the cytinder was
incubated for oie holr ar 65.C with aqilarion. Then 15 mt ot
prehybridizarion solution was removed tom the cylinder ano denatured
84