MaterialsNeeded

• 20mlglassscintillationvial
• Smallstirbar
• Foil
• Glycerol
• 1XPBS
• Pipets
• *P-phenylenediamine(EMDChemicalsInc.Cat#PX0730)
• Carbonate-BicarbonateBuffer(seebelow)

1. Wrapaglassscintillationvialwithfoilanddropinasmallstirbar.(PPDislight-sensitive.)
2. Witha10mlpipetadd9mlsofglyceroltothevial.
3. Withthe1000µlPipetmanadd1mlof1XPBS.
4. Placeonstirrerandbeginmixing.
5. Weighout10mgofp-phenylenediamineontheMettlerbalance.PPDistoxic.Wearglovesanddon’tinhaleit.
6. AddthePPDtothevialandstiruntillitisallinsolution(1-2hrs.).Themediumshouldappearalmostcolorlesstoaslighttintofyellow.IfitisanintenseyellowororangecolorthePPDismostlikelycontaminatedandwillhavebackgroundstaining.
7. pHthemountingmediumtoapHof8.0-9.0usingtheCarbonate-Bicarbonatebuffer.pHpaperofrange6.5-10.0shouldbeusedtocheckthepHofthemediumafteradditionof12dropsoftheCarb-Bicarbbufferandstirring.AdditionaldropsofbufferareaddeduntillthedesiredpHisreached.
8. Aliquotthemountingmediumandstoreat-70oC.

*FlakesofPPDarelargeandshouldbecrushedCarbonate-BicarbonateBuffer

1. Makeupa0.2Msolutionofanhydroussodiumcarbonate(2.12g/100ml)
2. Makeupa0.2Msolutionofsodiumbicarbonate(1.68g/100ml)

Take4mlsofA+46mlsofBandbringupto200mlswithDH2O.ThepHwillbe9.2.*Note:IfthePPDiscontaminatedorgoesbad(turnsabrowncolor)itwillstainDNA,soeachpreparationshouldbetested.Checkbylookingatmitoticcellstobesurethatchromosomesarenotstained.

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