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

AzE F: Cel Cvcle Cotuat at H4 eqe ftanscttoltan
c€nrriluged lor 30 second at 14,000 lpm (Eppendori microiLrge) to colect the
bacterial pellet. The supernalant was discsrdod and the ce|l p€llet was lysed in
150 /r or STEI bufer (50 mM Tis HCr pH 8-0, 50 mM EDTA, a% sucrose, 5%
Tdton). The conrenb ol the rub€ w6r6 boited exactly tor @ seco.ds a.d the
rub€s wer€ canrrituged for 15 minutss al room temperarure st 14,000 rpm io
collect tlra pellet. The pellgt which r6pr€sents bacterial debris co.taining
chrornosomal DNA, was removed with ast€r16tooth pick. Ths plasmd ONA ln
lho romaining lysate was prscipitatsd by addlng 200 /rl ol isopfopano and
micro-cenl.ilugaton ol the mixtule tof 10 to 30 minutes at 4'C. The pe l€l was
washed with 70% elhanol (1e, 10O &l was added to the pellet, and the lube
was spun 5 ro 10 minutes, Ihe pellst was at dried and the oNA dissolved
'or
in 40 rlolTE buffer.
ldentltlcatlon ot clones
Restriction endonuclsase dl9ssllon
To identify co.structs containing oligonucleotide inserls, plasmid
clones we.e digested wirh specilic restrictjon enzymes. Presenc€ ol lhe
oliqonucleoides cassetles would recreate a site lor Ava ll (lor all
mlranrs sxcepr GT.g) and increasa the size ot one ol rhe Taql
l.agments. Pst was used to conlirm thatrhe oligonucleotides contai.ed
lhe proper proxmal l€rmlnal soquences. Thereiore, lhe mini
preparations of pDNAs wefo individually digesied wilh lhe restricron
endonucleases Ava ll, Taql, Psl I and E@Rl. Typical .esviclion
endonuclease reaclions morained lhe desired plasmid DNA (4 /l) and