pH计校准的正常斜率范围为90%～110%。_
来自 : 蚂蚁淘
CSFExtractPrepforSpindleAssembly
ClaireWalczak11/95
ThisprotocolisessentiallyasdescribedbyMurray(1991),CellCycleExtracts.InMethodsinCellBIOLOGy,B.K.KayandB.Peng,eds.(SanDiego:AcademicPress),pp.581-605.I"veincludedaprotocolwhichemphasizesthepointsthatwefindaremostimportantforobtaininggoodCSFextractsthatarecompetentforCSFspindleassemblyandforcycledspindleassembly.Theindicatedbufferamountsaresufficientfora4frogprep.Forspermnucleuspreparation,seeaboveprotocol.Forspindleassembly,seeSawinandMitchison(1991)J.CellBiol.112:925-940.
NotesBeforeBeginning:ThequalityoftheeggsisessentialforgoodCSFextracts.Alwayssacrificequantityforqualitywhentryingtomakefunctionalextracts.Discardanybatchesofeggsthathave"puffballs"oractivatedeggsasmorethan10%oftheeggs.Weroutinelyuselaideggsandcollectatabout16-17hours.Ifyouaretryingtomakeextractsthatwillformspindlescompetentofanaphasechromosomesegregation,wefinditnecessarytouseonlyfreshlysqueezedeggs.Keeptheeggscool(16degCincubator)andonlybringthemtoRTrightbeforeyouarereadytopreparetheextract.
ThingsYouNeed
StockBuffers:
- 20XXBSalts:
- 2MKCl
- 20mMMgCl2
- 2mMCaCl2
- storeat4degC.
- 2MSucrose:
- Sterilefilterandstoreinaliquotsat-20degC.
- 1MHEPES,pH7.7:
- Sterilefilterandstoreat4degC.
- 0.5MK-EGTA,pH7.7:
- SterilefilterandstoreatRT.
ExtractPrepBuffers:
- MMR:
- 5mMHEPES,pH7.8
- 0.1mMEDTA
- 100mMNaCl
- 2mMKCl
- 1mMMgCl2
- 2mMCaCl2
- make2l-storeatRT.
- XB:
- 10mMHEPES,pH7.7
- 1mMMgCl2
- 0.1mMCaCl2
- 100mMKCl
- 50mMsucrose
- make250ml-makefresh.
- CSF-XB:
- 10mMHEPES,pH7.7
- 2mMMgCl2
- 0.1mMCaCl2
- 100mMKCl
- 5mMEGTA
- 50mMsucrose
- make250ml-makefresh.
- Dejellyingsolution:
- 2%cysteine;1XXBsalts,pH7.8
- waterto200ml-makewithin1hourofuse.
- EnergyMix:
- 150mMcreatinephosphate
- 20mMATP
- 2mMEGTA
- 20mMMgCl2
- 100ulaliquots-storeat-20degC.
Equipment
- 1600mlbeaker
- 1150X75mmpetridishes
- 5%gelatininddH20(at37!C)
- Flamepolishedcut-offpasteurPipettes(diameterofopeningapprox.2-3mm)
- LPC(10mg/mleachofleupeptin,pepstatin,chymostatininDMSO)
- CytochalasinD(10mg/mlinDMSO)
- 13X51mmultracleartubes
- SW55.1@16degCinultra
Procedure
BeforeStarting
- Getallsolutionsreadyandtubesintherack
- Havegelatin@37degC
- Coatpetridishwith100ul/dishofgelatin,swirlandreplacewithXB
- Bringfrogstoroomtempatthelastminute
Protocol
- Collectlaideggs:keepeggsinseparatebatchesifdistinguishabledifferenceinquality
- WasheggsinMMRtillallthecrapanddirtisremovedin600mlbeaker.
- Gardenawaythebadeggs(pickoutindividuallywithpasteur)
- RemoveasmuchMMRaspossIBLe.
- Dejellyin2%cysteinetillpacked(~5min)-removeallcysteine
- Washdejelliedeggs2-3XwithXBingelatin-coatedpetridish-removeallXB.Foreachwashswirltheeggsaroundthedishandthenlettheeggssettlebackdown.Theyshouldpacktightlyafterthejellycoatisremoved.
- Wash2-3XinCSF-XB(150mltotalvolume)-removeasmuchbufferaspossible.
- Wash2XinCSF-XB+105g/mlPIs(100mltotalvolume)
- Transferinto1mlofCSF-XB+PIs+100ug/mlcytochalasinDin13X51ultracleartubes(leteggsdropin)
- Suckoffallbufferfromtop(prettydry)
- Putintofalcontubeandspinfor10sec@#4inaclinicalcentrifuge.
- Removeallbuffer(prettydry)andputin1mlversilube
- Spinat#5for30secandfullspeedfor15secinaclinicalcentrifuge.
- Removeallbufferandversilube(asdryaspossible)
- Crush@16degC:15min@10,000rpm(fullbrake)inanSW55rotor.Wefindthatusingtheultracentrifugeatthisstepgivesmuchmorereproducibleextracts.
- Collectextractwith18gaugeneedlebypuncturingthesideofthetubeandgentlysuckingoutthecloudycytoplasmiclayer.Youshouldbeabletoobtainabout0.5-0.75mlofextract/tube.
- Add1/1000volumeofLPCandcytoD;1/20volof20Xenergymix;1/40vol2Msucrose.
ExtractisReadytogo!
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发布于 ： 2021-09-08
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