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

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agar plate used lor the initial wening ot the mgmbran€i the m€mbrane is
positioned race-up' (re, trrs bacl€rial sids is on lop) Ihe oiginal
plales w€re saved and incubated ove.n ght at room temPerature Io
regain bact€rial colonies.
Th€ membranes containing bacterial colonies were lysed dnedly on lhg
membrane disc by placidg il onlo whahan 3MM paper lor 5 minules,
whch was pre.soaked in 0.5 N4 NaOH solution. The m€mbrane disc
was n€utralizsd for 3 to 5 ninltes by placing it onlo another 3MM
Whahan sheet, which was saturated wilh a solution ol 1 M Tris/Hcl
{pH 7.5), The membrans discs were ina$ated tor an additonal 5
minures on anoth€r set ol3M[4 whahan sheets, saiuraled with 0.5 M
Tris-HCl pH 7 5 and 1.25 i,,l Nscl, and th6 discs wore air drled. Th€
DNA from th€ Lysed bacl€.ia was lired tothe membrane by Placlng the
disk under the UV,ighl tor 1 minule, and stored al 4'C in a sealed
plaslic bag. DNA {ragments spanning the inse.l was radiolabeled wilh
rhe PrimentrM Gndom prim6d oligolabeling kit (Stratagene) as
dessbsd by ihe manulaclLrr€r. lvlemblane discs wers pfe-hJ7bddized ln
25 ml of pre-hyb.idization solution containinq 5 X ssc (5 x ssc was
obhinsd from a 20 x ssc stock which contains 0.3 M sodium cltrale
and3 M sodium chloride, pH 7.25),5 x Denhaidl s soluion (lrom a 100
x srock conla.i.g 2% polyvinylpyrol done, 2% licol, 27o bovine serLrm
arblmin), O.O5 i.,l sodium phosphate bufier [pH 67i a 1 rr,4 slock'
solution ol phosphale buftff pH 6.7 is oblained by mixing 6€5 ml ol 1rM
40