Note:Allvolumesarecalculatedtocaterforfourplatesperpoint.

BaseAgar

1.Melt1%Agar(DNAgrade)inmicrowave,coolto40inawaterbath.Warm2XRPMI+20%FCSto40inwaterbath.Allowatleast30minutesfortemperaturetoequilibrate.

2.Mixequalvolumesofthetwosolutionstogive0.5%Agar+1XRPMI+10%FCS.

3.Add1.5ml/35mPetridish,allowtoset.Theplatescanbestoredat4forupto1week.

TopAgar

1.Melt0.7%Agar(DNAgradeagarose)inmicrowave,coolto40inawaterbath.(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(itisagoodideatoremovetheplatesfrom4about30minutespriortoplatingtoallowthemtowarmuptoroomtemperature).

6.Forplatingadd3ml2XRPMI+10%or20%FCSand3ml0.7%Agartoatube,mixgentlyandadd1.5mltoeachreplicateplate(usuallyplateoutintriplicate).NOTE:Onlydoonetubeatatimesothatagardoesnotsetprematurely.

7.Incubateassayat37inhumidifiedincubatorfor10-14days.

8.Stainplateswith0.5mlof0.005%CrystalVioletfor>1hour,countcoloniesusingadissectingmicroscope.