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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Nz F: Cel Ctd. Ctuol at h4 Cq. faBdiDtidl<br />
9r6y preclp tale devoloped, subs€quBnlly, ihe DNA mixlures we.e<br />
added b the mediud in th6 plalos in a drotrwis€ mann€r. Tho plates<br />
were gonrly swnbd and placed back in th€ incubalo. at 37'c lo allow<br />
the DNA to€rnerhe cells, Ths nsxt day, lhs c€lls w€re fel€d with 10 ml<br />
ol lresh OMEM medium. Folowhg 48 hours, the plates were washed<br />
twice with PBS phosphal€ bufier saine (PgS, 1 lil€r Pas was prepafed<br />
by mixing 0.2 g Kcl, 0.2 q KH,Po.,8 q NaCl. 1.14 g NaHrPO4' the pH<br />
was adjust€d to 7 4 and the sol! on was st6riliz€d by using 0 22 ,rm<br />
i lre4. The csns were hatuosted by aspnaling the medium, adding 1 m<br />
ot PBS and scraping lhe plates with a rubb6. polic€man. cell<br />
suspensions we.e lranslerr€d to 1 ,5 ml c€nlriluge tub€s The cells wsre<br />
subjected to cenrilugalion lot l minqtg at 14o0o rpm and the<br />
sup€rnatant was aspnai€d. Th€ p€lle|s were sav€d a.d slored al -<br />
m'C, o. processed lhe same dav lor chloramphenicol<br />
acevrransr€rase (caT) assays.<br />
DEAE-O6xlran M€lnod<br />
ROS 1712.8 and secondary ROB calls wsrs translgct€d with the DEAE<br />
dexrran m€thod (as desdibed by Ausubel, €4a1 1987) One dav prior<br />
to rhe rransfection, Ros 1712.4 cells w6re se€d€d d a densily or<br />
o.4r1G c€lls/1()o mm tlssue culture plal€ in HAMS F12 media<br />
supplemenled wnh 5%i€lalcafi serun, 100 un't/ml penicillin 1o0 !g/ml<br />
st€promycin, and 2 mM L-glutamins (kom GlBco)i nde het Ros and<br />
FOB cells r€quke a gap ot at l€ast 12 _ 14 hours alt€r plaung ror proper<br />
69
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