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

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Preparation ol glycerol stock badlorlal strairu lor slorage
sacterial cullurcs 0O mD Mr€ prepared by inodla on tom a singl6
colony or trom a previous glycerol stock (25 _ 50 pl) and ovsrnighl incubalion
at 37'C wlh shaking. Ihe nexl morning 500 rlol g.own bacteria were mxed
{fth 5oo pl ol sterile 8o% glyc€rol or 1oo % glycerol in cryotubes. The
suspefslons wer€ mxed by inv€rting th€ tubes mulliplgllmes and stored al _
70,c.
To ensure the clrr€cl idsntily ot plasmld DNAS grown Jrom thess
bacrerial stocks, construcls wer€ analyzed by both lestricron enzyme
analyses and seque.ce.analysis ol large scale Plasmid preparalions derived
lromlheslocks
large scare prepa.ation ol Plasmid oNA by alkallne lvsis
Large scale prepration of plasrnid DNA was initial€d by prepanng a
loml ove.night cultwe which was grown io late log phase to an optical density
or O.O al A@. The culll]re was lransloned to 5OO ml TB medium Oeriiic Brcth:
Ausubeler a/., 1987); the lquid culure was s€t !p in such a way that the arto
liquid ratio was appronmately 3:1 in each culture llask (reqardless ol size)
Cuhu.es were lncubated at 37'C ov€rnght in a shaking incubator with 10
mg/nlampicilin. Bacteria we.€ hatuesled by spinning samples in a sorvall
GS3 roror at 4'C ior 10 minutes at 5000 rpm.
Bacler alpellets wers dissolved in ao d ol ST€ solution (0.1 M NaCl, 10
mM T s HCl, pH aO, 1 mM EDIA PH 8.0). Tubes we€ centrillrged lo corlect
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