FreezedownGuidelines

WhattoSave

Foreach96-pack,eachlabwillhaveyeastrepresentativesfromallfourstrains:BY4741(orBY4730),BY4742(orBY4739),BY4743,andthehomozygousdiploid.Itisrecognizedthatthesefourcollectionswilleachhavegaps,dueto(a)somegenesbeingessential,(b)failuretoobtaintransformantsthatpassedall5PCRconfirmationtests.

CentralStorage:

Wewillbestoring4setsofAisolatesin2-mltubes.ForessentialgenesonlywewillstoretheBisolatesinadditiontotheAisolates.TwosetsgotoResearchGenetics,oneeachtoStanfordandEUROSCARF.

Duplicatesof"A"strains,"B"isolates,etc.maybesavedaccordingtoeachlab"spreferences

Sendstrainsoffwhenyou"redonewiththe96-packandhaveenteredallthedataforthosestrainsinthedatabase.

Storagein2-mltubes:

• PatchstrainoutonaYPD-G418plate.
• Color-codethecapsoftheSarstedttubeswithaVWRpenorequivalent--BY4741orBY4730-TheMATahaploid-REDBY4742orBY4739-TheMATalphahaploid-BLUEBY4743-Theheterozygousdiploid-BLACKThehomozygousdiploid-GREEN
• Pickyeastupwithatoothpickorimplementofyourchoiceandtransferto1.0or1.5mlYPD/15%glycerolinaSarstedttube.TransferenoughyeastintothetubetogetanOD600ofabout5(estimatebyeye).
• Freezeat-70°Cbeforecellsthesettle.
• One100placeboxshouldbeusedperbackgroundper96-pack,regardlessofthefactthattheboxeswillhavegaps.
• PuttheBisolatesoftheessentialgeneseitherinaseparateboxoratthe"end"ofthehomozygousdiploidbox.
• Sendondryice.
• Notifythepeopleyou"resendingthemtobye-mail

Tubes:2-mltubeswithwritingareathatfit100/2"box(Sarstedt#72.694.006)

Labels:Youwillreceiveprintedoutlabelsfor6setsoftubesfromStanford.Ifyoustartfreezingbeforetheseareavailable,labeleachtubewiththerecordnumber,strainname,batchand96-packco-ordinateontheside.

Boxes:NationalLabNet,9085492100,cat#R8300