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

A2iz F Cett cycta contat al Hl Gene I'ansciPtiah
0.5 to 1.0 mnr. Clones $€f€ eith€t Picked and dirsctly Prccessed ior
min|pbsrnd prcparadons, or itanslgr€d b a solid suppo'l
(nilrocellulose membran€ discs) and lysed in situ as a pr€Ude lo
colony hybridizaton rcp.€ss s a laPid and otlectiv€ technique lo
rsorato and id€nfry rccomblnant plasmids grown on LBlanljbiotic plal6
by lransf€r of bacierialcolonieg lo nilrocsllulos€ inembranes (Hanahan
ard M6s€lson, 1943). Alt matsials 4d solutions were aLnoclaved
beto.e uss. Nitrocellulose m€mbranes were wrapp€d in duminum foil
and auloclaved ior 15 cyclos. Ligstion mixturas wsrs lransformed inlo
DHsd and spread on LB/anlibiolic agar plal€s. Th€ pLates w6rs
incubated overnig al37'C i.rntilthe colonies were grown 0.5 mm in
diameter and removed from rhe incubabr'
Nitrocellulose membran€ discs were wotred using lorceps by placing it
over LB/anllboric agff plates wLthoul colonles Ths discs wer€ lilt6d
and gently placed over a second aqar pLat. containing baclsriar
colonies. fte membanes wers slighlly pressed to albw rhe colonies to
adhsrc lo th€ d scs. The postof ol lh€ membrane drscs were ma {eo
by pu.clut lng aneedlohrough bothlhe membranaand the agar' Ths
is idporlant tor orientng lhe membrane and lutu.e idertification ol lhe
positavB colonles. The memb.ane which now conlains a replica ol lhe
original bacleria colonleswas removed and temporarily slored onto the
39