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

tniz F: cen c\tto contat ot H4 G@ I@.ttott@
cenlrilugation. The bacteria wsrs resuspended in 10 ml ol CaCl2 bulfer
per rub€ and chilled on ic€ tor 30 minLtes. compot€nt c€lls were
co eded by cenr lugation as dessbod above and rosLrspendsd n 2
mloicacl, bulier pertub€. Aliquors (10o,,1) ol the compstent bacteia
wer€ transleiied into approximatsly 40 Eppendort tubes (1.5 ml) and
f.ozen down in liquid nit ogen al -70'C.
Baclerial translormatlon
Ligalon rnixtures wer€ lransiormed into DHsd by slendard merhods
(Manlatis el a/. 1942).In bri€t, approximately 100 rl ol competent cells
were lhawed on ice and 25 ng of plasmid DNA c 40.80 ng ol igated
oNA mixt!rc was mixed g€nlly and incubaled on ice ior 30 minutes.
The tubes were then transteiied ro a wat€r bath s€l at 42 0'c for
exactly 90 second lo heat-shock lhe cells. Subsequently, 1 ml LB
medilmwirhoLt ampicillin was added and lhe tubes were incubaled al
37'c tor 30 minlrtes whte shaking. For bacteria translormed with
igation mxrures, tlbes wer€ cenkfuged lo. 10 second 10 colect lha
pellgls These pe leG were roslspended in 1 /
10 ol ihe oflg nal volume,
and ih€ resultlng bacterial suspensions were sprsad on ampicilln
(anp) conhini.g LB agar plares. For the cornrol tanstornations w'th
unrr6at6o c.rc-ldr plasm,d oNA (whEh translorns fud' morP
elilcienrly) 50- i 0o p oi rhe or ginal ranslormallon rnixur€ was spread
overamp/LB ag plares. Plates were incubated in an invened position
ar 37'c lor 16 ro 20 hr, lesulring in bacterial colonies wilh a diamete. ol
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