Note:Allvolumesarecalculatedtocaterforfourplatesperpoint. BaseAgar 1.Melt1%Agar(DNAgrade)inmicrowave,coolto40inawaterbath.Warm2XRPMI+20%FCSto40inwaterbath.Allowatleast30minutesfortemperaturetoequilibrate. 2.Mixequalvolumesofthetwosolutionstogive0.5%Agar+1XRPMI+10%FCS. 3.Add1.5ml/35mPetridish,allowtoset.Theplatescanbestoredat4forupto1week. TopAgar 1.Melt0.7%Agar(DNAgradeagarose)inmicrowave,coolto40inawaterbath.(Itisimportantnottoexceed40,otherwisecellswillbekilled).Alsowarm2XRPMI+20%FCStothesametemperature. 2.Trypsinisecellsandcount.Itisveryimportanttohaveapositivecontrolline(eg.rastransformed). 3.Yourequire5,000cells/plate,thereforeyouneed20,000/tube.Adjustcellcountto200,000cells/ml. 4.Add0.1mlofcellsUSPensionto10mlyellowcappedcentrifugetubes. 5.Label35mmpetridisheswithbaseagarappropriately(itisagoodideatoremovetheplatesfrom4about30minutespriortoplatingtoallowthemtowarmuptoroomtemperature). 6.Forplatingadd3ml2XRPMI+10%or20%FCSand3ml0.7%Agartoatube,mixgentlyandadd1.5mltoeachreplicateplate(usuallyplateoutintriplicate).NOTE:Onlydoonetubeatatimesothatagardoesnotsetprematurely. 7.Incubateassayat37inhumidifiedincubatorfor10-14days. 8.Stainplateswith0.5mlof0.005%CrystalVioletfor>1hour,countcoloniesusingadissectingmicroscope.
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