Spotted Microarray Hybridization and Scan

• Mixtwocolorsamples(forgenomicDNA,Normally0.5ug),add2mlBlockMix(10mg/mlsalmonspermDNA(BRL)+4mg/mlyeasttRNA)
• UseSpeedVectodrythesampletoasmallvolume,normallylessthan5ml,doNOToverdry.
• Add50mlSigmaArrayHyb^TMHybridizationBuffer
• Heatmixtureto100°C5minutes,mixbypipettingupanddownifnecessary
• Spinfor5minutesat14,000RPMtopelletanyparticulatematerial
• PlacetheMicroarray(s)inahybridizationchamber(TeleChemorGeneMachines)(Topreventarraysfromdrying,sealarrayinhybridizationchamberwithWhatmannpapermoistenedwith100mlofH₂O.)
• Carefullytransfertheprobesolutiontoanewtubeavoidinganypelletedmaterial.
• Carefullypipettethehybridizationmixontothearray(betweenmarksetchedonbackofarray)
• Overlaywithacleanglasscoverslipbeingcarefultoavoidbubblesunderthecoverslip
• Sealchamberandplacein60°Cwaterbath.Incubate18-20hours

• Drytheexteriorofhybridizationchamber
• CarefullyopenhybridizationchambertoavoidH₂Oenteringthechamber.
• Removeslidefromthechamberandquicklyplacetheslide,arraysidedown,inacontainerfilledwith0.2XSSC,0.03%SDSandaslideholder.Positiontheslideupsidedownandataslightanglesothecoverslipfallsawayfromtheslide.Keepslideinthispositionuntilthecoversliphasfallenoff.�Whencoverslipfallsoff,transfertheslidetotheotherwashchambercontainingwashbuffer1andincubatefor5minatroomtemperaturewithgentlemixingonanorbitalshaker(speed3-4).
• TransferONLYtheslidetoacontainerwith0.2XSSC(filtered)andaslideholder.Incubatefor5minatroomtemperaturewithgentlemixingonanorbitalshaker(speed3-4).
• Transfertheslideholderwiththeslidetofresh0.2XSSC(filtered)andrepeatwash5min.
• Transfertheslideholderwiththeslideto0.05XSSC(filtered)andwashfor45sec.
• SpininslideholderseatedinaPlexiglasscarrieronmicrotiterplatecarrierfor5minutesat500RPMtodryslide.
• Scanslide.

ScanSlides

CurrentlyweusePackardScienceSA5000scannertoscanourmicroarrays.toobtaingoodsignalandlowsignal/noise,differentPMTsettingmightbeused.However,tocompareprobequalityandhybridizationquality,itissuggestedtoscanallslideatthesamepowerandPMTsetting.Nowweusethesetting:

• ForCy3,whichnormallyistheRNAchannel,Power85,PMT75.
• ForCy5,whichnormallyisthegenomicDNAchannel,Power85,PMT70.

HybridizationChambers	Telechem	Cat#AHC-1
ArrayHybBuffer	Sigma	Cat#A-7718