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Pol III whole cell yeast extract (small scale)
蚂蚁淘
/发布时间:1628271002 /浏览量:236
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.
SolutionsneededforWCE:
ExtractionBuffer(500ml)
| 100mMHEPES,7.9 | 11.9gHEPES |
| 245mMKCl | 9.14gKCl |
| 5mMEGTA | 10ml0.25MEGTA |
| 1mMEDTA | 2ml0.25MEDTA |
| H20to500ml |
Justbeforeuse,addproteaseinhibitorsandaddDTTto2mM.
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
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