ThisCGHProtocolisusedforDNAofgoodqualitywhenavailableinsufficientamounts.Weusuallydoreplicatehybridizationsusingsampleslabeled"inversely"(reversingthelabelfortestandnormalDNA"s).Ifappreciableartifactoccurs,thenalternativelabelsaretried. CotDNAvariesfromlottolot.WehaverequestedthatasingleLotbeputonreserveforouruse.Wethenmeasureitsconcentration(1mg/ml)andtestitforCGH. TheamountofprobeDNAusedcanvary,andshouldbeadjusteddependingontheintensityoftheproduct.Wegenerallyusethefollowingvoumesofthe~1ug/50ulnicktranslationproduct: FreshDNA:12ulParaffinDNA:45ulPCRproductfromfreshdna:20ulPCRproductfrommicrodissectedDNA:45ul ProbesizeisprobablythemostimportantaspectofCGHsuccess.ForgoodqualityDNA(fromfreshtissue),sizeshouldbeadjustedbyrepeatingthenicktranslation.Itisbesttouseprobeswhichareasmearbetween0.3-2.3kb.Sizecanbeadjustedbyreducingorincreasingthestandardtimefornicktranslationfrom60mins(using2.5-5ulofDNAse/Pol-1enzymemix.Paraffinsamplesalsoshouldbethissize,althoughoftentheyappearbigger.PCRproductsareusuallysmaller,rangingfrom0.1-1.5kb. ThequalityoftheslidesusedisthesecondmajorvariableinCGH.Eachnewbatchshouldbetestedunderdifferentconditions,suchasadjustingdenaturationtimeandtemperature. StoragetimeisanothervariablewhichaffectstheCGHquality.Aftersometimetheymaygivevariableresults.Thisiswhyitisimportanttorunacontrolsample.Usuallyifthechromosomebandingisverygood,thecghmaysuffer.Youmayneedtosacrificethebandingslightlyinordertogetoptimumcgh.CGHofDIRECTLABELEDTESTDNAvsNORMALDNA
DAY1:
1)ReprecipitateDNA"s
2)Denatureslides:
3)Hybridization:
DAY2:WASHESANDSTAINING
NOTESONCGHVARIABLES:
Cot-1DNA:
ProbeDNA:
Probesize
Slides:
Slidestoragetime