MikeSchultz,PavelAprikian,SteveHahn lastmodified Grow2litersyeastinYPD(with3%glucose)toanA600of1.5to2.0(inoculate~1.5mlsaturatedovernightin1litermediaandgrow~14hrat30degrees).Expect5-6gofcells. Harvestcellsandwashcellsoncein30mlcoldH2O. Washcellsoncein30mlcoldextractionbuffer. Weighcellpellet. Scrapepelletintoa10mlsyringeandsquirtintoa50mlscrewcaptubewhichissUSPendedintoliquidN2(thetubecanalsocontainalittleliquidN2ifdesired).Thiscellpelletcanbefrozenat-70deg.foratleastoneyearwithnolossofactivity. Precoolalarge(~8inchdiameter)mortarandpestle.Putondryice.AddalittleliquidN2tocool. Dumpinfrozenyeastandgrindbigpiecesuntileverythingisuniformlysmallpowder(~1min). AddalittlemoreliquidN2beingcarefultonotmakepowderflyallover. Grindagainforabout1min.RepeatliquidN2additionand1mingrinding. Useaprecooledspatulatoscrapethepowderintoaprecooledweighboat.Dumpintoa50mlconicaltube.Storetubeondryiceifgrindingotheryeastcells. Afterallgrindingiscomplete,transfertubetoiceandadd15%thetotalvolumeofextractionbuffer(i.e5gcells+750ulbuffer)andletsitonice20min.ScrapetheyeastsorbetintoanultraclearSW55tubeandspininultracentrifuge100,000xgfor1hrat4deg.(33,000rpminSW55). Afterspinning,~1/3volumeiscellpellet,~2/3volumeaclearyellowliquidandathinwhitelipidlayeronisontop.Removeclearliquidwithasyringebeingcarefultoavoidthepelletandlipidlayer. Dialyze3X1hrat4degreesvs500mldialysisbuffer. Aliquotandfreezeextracts. Expect20-30mg/mlprotein.Typically,use20microgramsinaPolIIIinvitrotranscriptionreaction. Justbeforeuse,addproteaseinhibitorsandaddDTTto2mM.SolutionsneededforWCE:
ExtractionBuffer(500ml)
100mMHEPES,7.9 11.9gHEPES 245mMKCl 9.14gKCl 5mMEGTA 10ml0.25MEGTA 1mMEDTA 2ml0.25MEDTA H20to500ml DialysisBuffer1.5liters:
20mMHEPES,7.9 7.14gHEPES 100mMKCl 11.2gKCl 5mMMgCl2 7.5ml1MMgCl2 1mMEDTA 6ml0.25MEDTA 20%glycerol 300mlglycerol H20to1.5liter
Justbeforeuse,addproteaseinhibitorsandaddDTTto2mM.
ProteaseinhibitorsandDTT:
0.1MPMSF(100x)
16mg/mlEthanol
Storeat-20degrees
0.2MDTT
32mg/mlH2O
Storefrozenat-20degrees
Benzamidine(100X)
31mg/mlH2O.
Storefrozenat-20degrees
Leupeptin(500X)
0.15mg/mlEthanol.
Storeat-70degreesforlessthan6months
Pepstatin(200X)
0.28mg/mlmethanol
Storeat-20degrees.
Chymostatin(2,500X)
5mg/mlDMSO
Storefrozenat-20degrees
