CGH of Biotin Labeled DNA vs Digoxigenin Labeled DNA

DAY1:

1)ReprecipitateDNA"s

• AddthefollowingDNA"stoa1.5mlcentrifugetube,mixingwithpipet:20ugCot-1DNA(~20ul)~200ngBiotinlabeledDNA(~10ul)~200ngDigoxigeninlabeledDNA(~10ul)
• Add1/10thvolumeof3MNaAcetate,mixingwithpipet.
• Add2.5X(original)vol100%EtOHtopptDNA,vortexgently.
• Spin30minsat14Krpm,4C.
• Decantsupernatant;blotdry,beingcarefultoavoidDNApellet.
• Add10ulofMM1/H20mix(70%MM1/30%H20).
• Carefullydissolvewithpipet,andgentlyvortex.
• Quicklyspin(1sec)tobringvolumetobottomoftube.

2)Denatureslides:

• Selectslideswithspotsinagoodlocation,plentyofmetaphases,andlittleornocytoplasmaroundthemetaphasesornuclei.
• Markeachspotonthebackoftheslidewithadiamondpen.
• Prewarmslideon37Chotplatefor1minute.
• Denatureprewarmedslidesin70%formamide/2XSSCfor2.5-10minsat73Cinsideacoplinjar(timewillvarydependingonslides).
• Denaturenomorethan3slidesatatime.
• Dehydrateslidesthrough70%,85%,and100%ethanols,2minseach.
• Wipebacksofslidesandairdryuprightonkimwipes.

3)Hybridization:

• Placeslideson37Cwarmer1-2minutesbeforeaddingprobes.
• Denatureprobemixat70-75Cfor5mins(optimumtempwillvarywithslides).
• Applydenaturedprobeimmediatlyontowarmedslideonthehotplate.
• Coverslip(18mm)andsealwithrubbercement.
• Letrubbercementdry5minsonwarmer.
• Incubatefor2-3daysat37Cinahumidchamber.

DAY2:WASHESANDSTAINING

• Removerubbercement.Slidecoverslipsoffgently.
• Wash3Xfor12minseachat45Cin50%Formamide/2XSSC.
• Wash1Xfor10minsat45Cin2XSSC.
• Wash1Xfor10minsatRTin2XSSC.
• Removeslide,quicklyblotback(toremoveexcessliquid).
• Add85ulof4XSSC/1%BSApreblocksoln(keptinaliquotsinfreezer)andgentlycoverwitha24X50mmcoverslip.
• Incubatefor5mins,thengentlyremovecoverslip.
• Add85ulofamixofanti-digoxigenin-rhodamine/FITCAvidin(at1:200/1:400inpreblocksolution;e.g.2ulanti-digrhodamine+1ulFITCavidinin397ulof4XSSC/1%BSA).
• Gentlycoverwitha24X50mmcoverslip.
• Incubatefor45-60mins(protectingfromlight).Gentlyremovecoverslip.
• Wash10minseachatRTin4XSSC,then4XSSC/0.1%Triton,then4XSSC.
• Rinseslides2XinddH20for5-10minseach,airdryupright.
• Apply8.0ulof0.2ug/mlDAPIinantifade(22mmcoverslip).
• LetsitatRTforatleast2hours.DonotcollectimagesthesamedayasDay2staining.

NOTESONCGH:

Amplificationofindirectsignals:

IfFITC-Avidinhybridizationisverydim,butsmooth,thesignalcanbeamplifiedusingourstandardbiotinamplificationprotocol.However,ifitisdimandgrainy,thesignalwillonlygetbrightandgrainyandtheCGHprofileswillbetoonoisyforinterpretation.

Cot-1DNA:

CotDNAvariesfromlottolot.WehaverequestedthatasingleLotbeputonreserveforouruse.Wethenmeasureitsconcentration(1mg/ml)andtestitforCGH.

ProbeDNA:

TheamountofprobeDNAusedcanvary,andshouldbeadjusteddependingontheintensityoftheproduct.Wegenerallyusethefollowingvoumesofthe~1ug/50ulnicktranslationproduct:

FreshDNA:12ulParaffinDNA:45ulPCRproductfromfreshdna:20ulPCRproductfrommicrodissectedDNA:45ul

Probesize

ProbesizeisprobablythemostimportantaspectofCGHsuccess.ForgoodqualityDNA(fromfreshtissue),sizeshouldbeadjustedbyrepeatingthenicktranslation.Itisbesttouseprobeswhichareasmearbetween0.3-2.3kb.Sizecanbeadjustedbyreducingorincreasingthestandardtimefornicktranslationfrom60mins(using2.5-5ulofDNAse/Pol-1enzymemix.Paraffinsamplesalsoshouldbethissize,althoughoftentheyappearbigger.PCRproductsareusuallysmaller,rangingfrom0.1-1.5kb.

Slides:

ThequalityoftheslidesusedisthesecondmajorvariableinCGH.Eachnewbatchshouldbetestedunderdifferentconditions,suchasadjustingdenaturationtimeandtemperature.

Slidestoragetime

StoragetimeisanothervariablewhichaffectstheCGHquality.Aftersometimetheymaygivevariableresults.Thisiswhyitisimportanttorunacontrolsample.Usuallyifthechromosomebandingisverygood,thecghmaysuffer.Youmayneedtosacrificethebandingslightlyinordertogetoptimumcgh.