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

a2lz F: celt cyd. c.ntol d H4 G@. fansqbtbn
acevlt.ansferas€ (CAT) g€n6. Thls CAT lragm6 was inserted inlo the
irnermediate constructs to yield th6linal mutant H4 promoler/cAT conslructs-
Ligation reacrions we.o set up by adding lhe vector and Lnsert f.agmenls d a
rnolar ralio oi 1r5 (vectoiinsed) tor cofstruction ol ihe inlerrnsdiate plasm ds
and 1:3 tor ligalions lnvolving rh€ cAT fragment and the intermediale mliant
clnslrucl- Feaction were iniliated by addition ol 1 unil of T4 ONA ligase in a 20
rl reacrion vollme which includes 1 X ligase butfer (50 mM HCIpH 7.6, 10 mM
Mgc2, 10 mM DTT (dthlothretol), 1 mlM ATP,50 pglml BsA, bovlne serlm
alblmin). Each Ligation mixtu.e conrained a totalof r00 n9 oNA. To check ior
lhe p.esence ot recombinant DNA Lagmenr, as weli as lhe efecliveness of the
ligalion reaclion, 5 rlol ligalion mixtur€ was loaded on a 1% agarose gel.
oNA rransformallon inro E col/ baclerla
Frepa.arbn ol compelenl callr
A l0 ml culrlre of e co, DHSd was grow. wirhout ampicillin by
inclbaron overnighl ln a shaking inclbator. A sampls oi the culture (2
ml) wasinoculaied nro 1@m oiLB medium in a side-arm flask wilhout
anpicillin. To monito. bacleial Eowth, tr€ optical densily ol the culture
was determined d 30 minur€ ideruals until lh€ c1rllure reached a
densry of 0.400 A@. Bact€ria cultufes we.e vansl€fied into lwo 50 rnl
conicsllub€s, and chill€d on lc€ lor 10 minules. Bacrerial pellers woro
collected by c€nritugalion ar 2!0o rpm for 5 minutes at 4'c in an lEc
rotor. Each p€llel was rssusp€odsd in 10 ml ol CaCl2 bufier (60 mM
CaCl,, 1s% glyc€rc|, 10 mM Pipes) and again pe leled by