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

aziz F: ce dde c@no| ol H4 Gqe fanscdorbn
pipe e. Tho Manifold il was plsc6d on top ol th€ m€mbrane and the
davice was ciosed at both sid€s using plaslic closure pieces (provd€d
wilh the apparatus io the boi)- The apPaatus $as hooked up lo th€
regular laboratory vacuum lines. and vacuumwas appled careillly Ths
wells were nsed wilh 1 M NH.OAc and 400 rrl ot sample was loadsd ln
eaci slot, slots wers washed with lM NH.oP€ again. Ths danitold w€s
femoved carelully and lhe nylon m€mbran€ was air dri€d The DNA
was nxed by placing the zeta probe membrane under the UV llght for 1
minlre andthe membrane was stored in aseal€d dasric bagal4 C
BIors were prehybidi2ed in cylind€ls using 25 ml hybridianion solulion
(12,5 mM sodum pyrcphospfral€, 1 lv Nacl,20 mM Tris pH 7.6, 2 mM
EDTA pH 7.5, O.l% SDS, 10 x Denhardt's solulion and 250 rglml
lr4hly denatured Bdom oNA lrcm E colt or salmon spem)-
Membranss were incubaled at 65'C lor 2 lo 4 hou.s Blots were
hybrdzed n 3 mioi hybidizauon solullon as above wilh the addition oi
2.0 to 2.5x1Od TCA precipilabls counts of radiolabel ed FNA transcripls
Hyb.idization was performed by incubaton st 65'C lor 72 hours Elols
were washed wilh shaking at 65'c lor 30 minlt€s ln a solunon
cootaining 2 x ssc and 0.1% qDs. This slep was repealed two rLm€s
The membran€ was washed lwics with another solution (containing 0.2
x ssc and 0,1% SDS) with each wash pertormed tor 30 minltes at
65'c. Flnally, ths blots wero wash€d once wilh 0 1 x SSC/o1% Sos
lor 20 minul€s at 65'C.
104