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Freezing Worms
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FreezingWorms

byMichaelKoelle

4/6/94

I.Thepreferredmethod

1.Washwormsoffof1largeplateor3smallplatesin3mlsSBasalintoasterile15mldisposablecentrifugetube.WormsshouldbeharvestedoffofjuststarvedplateswhicharepredominantlyL1andL2.Thisshouldbe1dayafterthebacteriahavebeenexhausted.Thewormbooksaysdauersdon"tfreeze,butE.Jorgensonsayshefreezesfromreallyoldplatesanditworksfine.Rumorhasitthatiftheplatesaren"tcompletelystarved,thefreezingwon"twork-i.e.foodinthegutsomehowpreventsthewormsfromsurviving.Towashthewormsoffplates,add~2mlsSBasaltoeachsmallplate(w/sterile5mlglassPipetteandapasteurpipettebulb),swirlbrieflytodislodgeworms,andsuckoffthesUSPensionwiththesterileglasspipetteandplaceintoa15mlsterileplasticcentrifugetube.

2.Spindowninaclinicalcentrifugefor~30sec.Removeallbut1.5mlofthesupernatant.

3.Add1.5mlfreezingsolution,mixwell,andaliquot1mleachinto3sterilefreezingvials(NuncCryoTubes#363401).

4.Freezeslowlyto-80deg..Thisisaccomplishedbyplacingthevialsinastyrofoamrack(thekindthat15mldisposablesterilecentrifugetubescomein),placinganotherinvertedsuchrackontopofthefirst,fasteningthetworackstogetherwithrubberbands,andplacingina-80deg.freezer.

5.Thenextday,movetwovialstoapermanentlocation.Recordthestrainnumber,genotype,andcommentsinacomputerdatabase.

6.Thethirdvialshouldbeusedforatestthaw.Takethevialoutofthefreezer,thawquicklybyholdinginyourhandorina37deg.waterbath(leaveinthebathonlyuntiltheiceisgonesoasnottoheatitup).Candumpthewholevialoutinaseededlargeplate,oruseasterile200ulpipettemantiptowithdrawthebottom200ul(containingthesettledworms)andplaceitonasmallplate.Thenextday,picklivewormstoanewplate.

II.Theagarmethod.ThisgivesmuchlowerviABIlitythantheabovemethodinmostpeople"shands,buthastheadvantagethatonedoesn"tneedtothawanentirevialatatime.Onbalance,Idon"tthinkitisworthriskinglosingstrains(especiallyriskywithheterozygousmixtures)togetthisminorsavings.However,somepeopleseemtohavebetterluckwiththismethodthanme.

1.Washwormsoffof1largeplateor3smallplatesin3mlsSBasalintoasterile15mldisposablecentrifugetube.WormsshouldbeharvestedoffofjuststarvedplateswhicharepredominantlyL1andL2.Thisshouldbe1dayafterthebacteriahavebeenexhausted.Thewormbooksaysdauersdon"tfreeze,butE.Jorgensonsayshefreezesfromreallyoldplatesanditworksfine.Towashthewormsoffplates,add~2mlsSBasaltoeachsmallplate,swirlbrieflytodislodgeworms,andsuckoffsuspensionwithasterileglasspipetteandplaceintoa15mlsterileplasticcentrifugetube.

2.Chillonice15"orlonger.

3.Meltfreezingsolution+agarinmicrowave(carefulltofullymeltbutnotboilover).

4.PutFS+agarbottlein50deg.waterbathfor~15".

5.Spinthewormsdown(30sec.inaclinicalcentrifuge),andremoveallbut3mlsofsupernatantwithasterilepipette.

6.Pipette3mlFS+agarintoatubeofwormsandimmediatelymixbyinversionoftube8-10X.

7.Pipette1.8mlofthemixtureinto3preparedfreezervials.

8.Repeatsteps6and7foreachstrain.

9.Freezeslowlyto-80deg..Thisisaccomplishedbyplacingthevialsinastyrofoamrack(thekindthat15mldisposablesterilecentrifugetubescomein),placinganotherinvertedsuchrackontopofthefirst,tapingthemtightlytogether,andplacingina-80deg.freezer.

10.Thenextday,movetwovialstoapermanentlocation.(Or,toaboxinthe-80deg.,andwhentheboxisfull,moveallthevialstoaliquidN2freezer).Recordthestrainnumber,genotype,andcommentsinacomputerdatabase.

11.Thethirdvialshouldbeusedforatestthaw.Caneitherthawthewholevialanddumponalargeplate.Or,usingaflamedspatula,scrape~0.1mloffrozenwormsontoasmallplate,andputtheremainderofthevialbackinthefreezer.

FreezingSolution

200ml1MNaCl

100ml1MKPO4,pH6.0

600mlglycerol

Bringto2literw/dH20

Distributeto200mlbottles;autoclave

Add0.06mlsterile1MMgSO4per200mlbottle

Freezingsolutionwithagar

freezingsolutionwith0.4gagaraddedper100mlandreautoclavedfor20min.

Sbasal

5.8gNaCl

50ml1MKPO4

950mldH20

1mlcholesterolin95%EtOH(5-10mg/ml)

swirltodisperse,andautoclave

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