Injection of C. elegans 实验方法
来自 : 蚂蚁淘
InjectionofC.elegans- lineupthree22x50mmcoverslips
- placedropofmolten2%agaroseoneach
- quicklyplaceslideontopoftheagaroseandwait20seconds(seefigure)
- carefullyseparatecoverslipsfromslidesothattheagarosepadremainsonthecoverslipanddry(overnightatroomtempor15minuteson65degreeheatblock)
**hint:pressthumboncoverslipsbeforedrippingtheagarose-theagarosetendstosticktothe"dirty"surfaceofthecoverslipinsteadoftheslide
- pullneedles(Narishigepullersettings:4/8/10)fromborosilicateglasscapillaries
- spindownRNApreptopelletanydebri(fullspeed5minutes)
- load0.5ulRNAontoendofneedleandallowittofillthetipbycapillaryaction
- mountneedleonmicromanipulator
- breakneedletipuntilRNAflowsoutofthetip
- placedropofheavymineraloilonpad
- pickup"younger"adultwithplatinumwirepick
- transferwormtodropofoilandallowthewormtotouchthepad(headortail)
- upontouchingthepad,thewormwillbegintostickandyoucanslidethepickoutfromunderthewormsothatthewormliesrelativelyflatandextended
- setupinjector:
- turnonmicro-injector
- openvalveonpressuretank
- openvalveofpressureregulator
- setinjectionpressures
- linepressure:approx4500hPa
- injectionpressure:900-1200hPa
- backpressure:80hPa
placemountedwormonscope(10Xlens)andorientthewormata30degreeangletotheneedledirectionswitchtohighermagnification(40Xlens)andbringgonadintofocus- honeycombpatternisthesurfaceview-lumenviewhasgranularmaterialinthecore
moveneedleintothesamefocalplanepressneedleintothegonad-thegonadwilldeformaroundtheneedletapsideofthestagesharplytocausetheneedletopuncturethegonadwall
- depresspeddletobeginfillinggonadwithsolution
- stopfillingwhengonadexpandsalongentirelengthbutbeforeit"pops"bydepressingpeddlesecondtime
- pullneedleoutofgonad
- clearstickydebrifromneedlebyapplyingline-pressuretocausesolutiontoflowfromtip
- injectothergonadinsimilarmanner
- placedropofegg-saltsonwormto"rehydrate"(wormshouldbeginswimming)andallowaboutfiveminutesrecoveryatroomtemp
- oncethewormisswimmingwell,transfertoOP50-seededplatewithmouth-Pipette
- passagewormatregulartimeintervalstodeterminetimingofonsetofRNA-interferenceeffects
- goodneedlesarethekey......andlargelydependentonyourluckwhenbreakingthetip
- breakneedlebygentlypressingtipagainst18x18mmcoverslipmountedondropofoilon22x50mmcoverslip
- ifthewormsmovetoomuchonthepadtrydryingthepadsonheatblockimmediatelybeforeusing
- candomultipleinjectionsonsinglepadbyusingsmalldropsofoilatmultiplespots:injectwormandrecoveritineggsaltswhilemountingandinjectingasecondworminneighboringdrop,whensecondiscomplete,transfertherecoveredfirsttoplate,etc....
- caninjectmultiplewormswithinthesamedropofoil-justdon"tdotoomanythatittakestoolongandthewormsdessicatebeforeyoucanrecoverthem
- recoverytimelongerthat5-10minutesusuallynotnecessary-evennon-swimmingwormswillrecoveraftertransferedtoseededplate
- Fire,A.,etal.,(1998)Nature391:806-811
- Mello,C.C.,etal.,(1991)EMBO10:3959-3970
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