FREEZINGWORMSTOCKS 1.Grow3-56cm.platesofwormsonNGMwithOP50untiltheplateisstarvedwithlotsofyounglarvae.TheL1saretheonesthatsurvivefreezingthebestandarethemostlikelytobefertileuponthawing. 2.Warmfreezingbuffer(FB)toroomtemperature. 3.LabelfiveCRYOSTORAGEtubesforeachstocktobefrozenwiththestrain"sID#andthedate.Capsiclesfitintothetopsofthecryostoragetubes.Labelboththetopandthesideofeachtube. 4.WashwormsfromoneplatetothenextwiththeFBusingasteriletransferpipettotransferwormsandbuffer.UseanewpipetforeachwormstockandbecarefulnottocontaminatetheFBstockwithworms. 5.Aliquota0.5ml.volumeofwormsintoeachoffourlabelledcryostoragetubes. 6.Putthetubesofwormsintoastyrofoamfreezingboxandputat-80Cforatleast24hours.Labeltheboxwiththedateandyourinitials. 7.Transfertubestoliquidnitrogentanksandfillouttheappropriatepaperworkforstoragerecords. 8.Thawatubetocheckifthewormssurvivedthefreezingprocess.Wormsmustberegrownbeforetheycanbefrozenagain(i.e.thesamewormwillnotsurvivebeingfrozentwice). FreezingBuffer: MixNaCl(2.93grs) KH2PO4(3.4grs) Glycerol(30%vol) NaOH(2.8mls). Taketo500mlwithdistilledwater. Autoclavefor20minutes. Allowtocooltoabout60C. Add3mlsMgSO4sterilely. Aliquotintosterile50ml.cultureflasks. Storeat4C.
