PreparationofCopyStandardsforPCRGenotypingPCRscreensmustbedesignedtodetecttransgeneDNAatthesinglecopylevel.Todemonstratethislevelofsensitivity,non-transgenictailDNAisspikedwithaknownamountoftransgeneDNAistoproducetransgenecopystandards.Thesestandardsshouldbere-runatthetimetailDNAsamplesfrompotentiallytransgenicanimalsaretested.

CalculationofCopyNumberStandards

Assumption:theHaploidcontentofamammaliangenomeis3X10⁹bpAssumption:youhave2microgramsoftailDNAavailable

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.

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