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

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Procedure

BeforeStarting

1. Getallsolutionsreadyandtubesintherack
2. Havegelatin@37degC
3. Coatpetridishwith100ul/dishofgelatin,swirlandreplacewithXB
4. Bringfrogstoroomtempatthelastminute

Protocol

1. Collectlaideggs:keepeggsinseparatebatchesifdistinguishabledifferenceinquality
2. WasheggsinMMRtillallthecrapanddirtisremovedin600mlbeaker.
3. Gardenawaythebadeggs(pickoutindividuallywithpasteur)
4. RemoveasmuchMMRaspossIBLe.
5. Dejellyin2%cysteinetillpacked(~5min)-removeallcysteine
6. Washdejelliedeggs2-3XwithXBingelatin-coatedpetridish-removeallXB.Foreachwashswirltheeggsaroundthedishandthenlettheeggssettlebackdown.Theyshouldpacktightlyafterthejellycoatisremoved.
7. Wash2-3XinCSF-XB(150mltotalvolume)-removeasmuchbufferaspossible.
8. Wash2XinCSF-XB+105g/mlPIs(100mltotalvolume)
9. Transferinto1mlofCSF-XB+PIs+100ug/mlcytochalasinDin13X51ultracleartubes(leteggsdropin)
10. Suckoffallbufferfromtop(prettydry)
11. Putintofalcontubeandspinfor10sec@#4inaclinicalcentrifuge.
12. Removeallbuffer(prettydry)andputin1mlversilube
13. Spinat#5for30secandfullspeedfor15secinaclinicalcentrifuge.
14. Removeallbufferandversilube(asdryaspossible)
15. Crush@16degC:15min@10,000rpm(fullbrake)inanSW55rotor.Wefindthatusingtheultracentrifugeatthisstepgivesmuchmorereproducibleextracts.
16. Collectextractwith18gaugeneedlebypuncturingthesideofthetubeandgentlysuckingoutthecloudycytoplasmiclayer.Youshouldbeabletoobtainabout0.5-0.75mlofextract/tube.
17. Add1/1000volumeofLPCandcytoD;1/20volof20Xenergymix;1/40vol2Msucrose.

ExtractisReadytogo!