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DNA labeling by nick translation

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DNAlabelingbynicktranslation

reagents:DNAforlabeling(concentrationc>150ng/µl)modifiednucleotides:Biotin-16-dUTP,Digoxigenin-11-dUTP,conc.1nmol/µl(BoehringerMannheim)dNTPs(regularnucleotides):dATP,dCTP,dGTP,0.5mMeach,dTTP0.1mMNTreactionbuffer10x(0.5MTrispH8,50mMMgCl2,0.5mg/mlBSA)b-ME(beta-mercaptoethanol)0.1MDNase(stocksolution3mg/ml)1:2000dilutedinaquabidest.Pol:KornbergDNA-polymerase5U/µl(e.g.BoehringerMannheim)EDTA(0.5M,pH8.0)SDS(20%)

foroneNTreaction5µgofDNAisused:

Mix(Vtotal=50µl):	1probe	mixforNprobes
NT(10x)	5µl	(N+1)*5:
b-ME	5µl	(N+1)*5:	formorethan1probe
dNTPs	5µl	(N+1)*5:	Pipette19µltothe
Bio/Dig-dUTP*	2µl	(N+1)*2:	DNA+H2O
DNase(1:2000)	1µl	(N+1)*1:
Pol	1µl	(N+1)*1:
---------------------	---------------------	---------------------
DNA+H2O	31µl
	=====
	50µl

*inthestandardprotocolTumorDNAislabeledwithBio-dUTP,NormalDNAislabeledwithDig-dUTP

Pipetteonice!

incubationfor2hrsat15°C-->putprobesonice-->test5µlofthemixinanagaroseelectrophoresis,optimallengthofDNAfragmentsshouldbebetween100-1000bp(inthemeantimestoreprobesat-20°C)-->ifneccessaryincubatelongerafteradditionofnewDNAseandPol-->add2.5µlEDTA(0.5M,pH8.0)and2.5µlSDS(20%)tostopthereaction,keeptheprobesat-20°Cuntilhybridization

Optimalfragmentlengthafternicktranslation

DNAafteragarosegel	===>	DetectionoflabeledDNAbyacolorreaction
electrophoresis		aftertransfertoanylonmembrane

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DNA&nbsp;labeling&nbsp;by&nbsp;nick&nbsp;translation

DNA labeling by nick translation