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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a2t2 F: cdr cw. ctuc ot <strong>H4</strong> 6.n. f@d4@<br />
raostered io ih6 37.c wat6r bath to. 15-20 minul€s tor annEati.g,<br />
Dunng |his lncubation, ths did€oxyructsotird€ l€mina$m mtes (2.5 /<br />
each ol ihi lour dille.enr basss, i6., dA! dT, dc and dc) werc added in<br />
small 5OO pl €ppendo.l tubss a.d tstt at rcom tgmp€€turc. |.;beting<br />
mir (provid6d in the kD was dlutod 1:5 wilh di3iitt€d tt, and 2 Et of this<br />
dilurion kas added in e8ch rnn€ating r.action 0o a totat votume ot 10<br />
pl). Each annoa,ing r€adion was turther 3uppi6h€ntisd with 1 / ot 0,1<br />
M OTT, r rlol lssl dATP {10 /ci/,lti sp€cilic acttity 1OOO Cilmmot)<br />
aod wilh 2 /l of a 1 t7 mixur€ of th6 s€qu6nas6 €nrym€ and saquenass<br />
dilution bufi€. (linal votum€: 14 /t), Aflar indbation room<br />
temperauro ror 5 minutssi 3,5 ,, ol €acn ,6action was add€d to !h€<br />
.ucr€olida spocilic tsrminarion mix€s (,,€,, cra, cIT, dc and dc mixrures)<br />
akeady aliquored. Tubas wer6 irct,bated tor an additionat 5 minutes<br />
and slopp6d by adding 4 /t ot gsl to8ding buf6r. AI samptes werc<br />
heared lor 4-5 minutes 6t 8o'c b€iorc toading on a 6%<br />
polyacrylamid6/0.7 M urca d6naturi.E gst. Th€ gat was c6st€d with a<br />
shark s toorh comb. ons hou bsfoG toading, lhe get was p€run lo.<br />
15-30 minul€s, and was run lor 2.2.5 hours a,r6. toadtng tha sampres<br />
(gr€crrophor€sB !m6 dep€nds on rh6 si26 ol DNA t agm€nts and the<br />
@ii6spondn9 s€qu6nc€s ro b€ Fad). IhE g€l was dried und€r<br />
vacuum and e4osed to Kod6l( i-oMAT AF film overnight and<br />
deveroped |he n6lt mo.ning (s€€ Fi$. 10, 11 €nd 12).<br />
48
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