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

M. F: Ce|| Crd. Cqnrot ot Ha Gerc TEneDiM
Prepararion ol radlo-labelled probe tor soulhe.n hyb.idization
Radiolabelled DNA probes lor South€tn hybddizallon werc prepared
by radom labelling. h b.iel, the 1.7 kb CAT insen iragm,eni derived
lrom pFO1o8CAT was radio]abded wllh the PrmeltrM randomly
prlmed ollqolabeing kil (Stalagene) as descrited by lh6 nafulacllrer.
h bri6i, 25 ng oi CAT ONA in 1 plwas mired rrith 10 !l ol random
oligonlcleotide primer, and ths reacrion mixturg was adjusred wirh 23 /
high qual'ty deionized and disl/tred H2O ro a lo|at votume ot 34 rt. The
tube was hear treaied lor 5 minules d 95. to 10O' in a beaker. The
rlbe was cenl.iluged brislly at room tempe.aiure 10 co tect ths
cond€nsed liquid on the cap ollhe tube The lolowing reag€nis were
added i.lho readon rube: 10 !tot 5 X dctp prtmer buti€r (conlatning
dATP, dGTP and dITP), 5 pl ot labell€d nucrsotids [4J,P] dcrp (10
/rcilpl - 3000 Cilmmot) and 1 /it or ExoC) ktenow enzy'ne (5 unir/ltt).
The conterns ot the lubes were mixed by pipene mixing. Afie. bdef
centritugarion, the samptes were incubat€d at 37 . C fo. i 0 minures. The
read'on was sropp€d by adding 2 pl oi stop mir (recip€ according to
uninco.porated nucleotides wero removed by psssing th€ p.obe
through Quick SpinrM columns. Eelore usir'g rrre spin cotLrm., the
unopened corumn was ihverted severall mes to mlx the G.25 s€phadex
beads in the blfter, The lop ld was removed tirst lrom lho c$ umn and
rhen th€ bonom iip, which is impo.lanr to maintain a smooth flow oi rhe
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