PreparationofCopyStandardsforPCRGenotypingPCRscreensmustbedesignedtodetecttransgeneDNAatthesinglecopylevel.Todemonstratethislevelofsensitivity,non-transgenictailDNAisspikedwithaknownamountoftransgeneDNAistoproducetransgenecopystandards.Thesestandardsshouldbere-runatthetimetailDNAsamplesfrompotentiallytransgenicanimalsaretested. Sincethetransgenicfoundermicearehemizygous: massoftransgeneDNA=NbptransgeneDNA1microgramgenomicDNA3X109bpgenomicDNA Example:fora5,480bptransgeneinsertorplasmid massoftransgeneDNA=5,480bpclonedDNAor1microgramsgenomicDNA3X109bpgenomicDNA massoftransgeneDNA=(5,480bpclonedDNA)X(1µggenomicDNA)or3X109bpgenomicDNA massoftransgeneDNA=1.83picograms Thus,topreparea1copystandard:add1.83pgoftransgeneDNAto2microgramtailDNA10copy18.3pg50copy91.5pg100copy183pg ForuseasaPCRstandard,usethesinglecopyspikedDNAasasubstratetotestthePCRassayyoudevisedforgenotypingtransgenicmice. ForuseinSouthernblotanalysis,digestthetailDNAasyouwouldforSouthernanalysis,andaddthetransgeneinsertDNA(nottheentireplasmid)justbeforeyouloadyourgel.RemembertoreserveonelaneforgenomicDNAonlywithnospike.ForanexampleofcopystandardsinSouthernblots,refertoCamperSA.1987.Researchapplicationsoftransgenicmice.Biotechniques5,638-650.Clickhereformorereviewarticles.CalculationofCopyNumberStandards
Assumption:theHaploidcontentofamammaliangenomeis3X109bpAssumption:youhave2microgramsoftailDNAavailable