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        DNA labeling by nick translation

        互联网

        2094

        reagents:
        DNA for labeling (concentration c > 150 ng/µl)
        modified nucleotides: Biotin-16-dUTP, Digoxigenin-11-dUTP, conc. 1nmol/µl (Boehringer Mannheim)
        dNTPs (regular nucleotides): dATP, dCTP, dGTP, 0.5 mM each, dTTP 0.1 mM
        NT reaction buffer 10x (0.5 M Tris pH 8, 50 mM MgCl 2 , 0.5mg/ml BSA)
        b-ME (beta-mercaptoethanol) 0.1 M
        DNase (stock solution 3 mg/ml) 1:2000 diluted in aqua bidest.
        Pol: Kornberg DNA-polymerase 5 U/µl (e.g. Boehringer Mannheim)
        EDTA (0.5 M, pH 8.0)
        SDS (20%)


        for one NT reaction 5 µg of DNA is used:

        Mix (V total = 50 µl): 1 probe mix for N probes  
        NT (10x) 5 µl (N+1) * 5 :  
        b-ME 5 µl (N+1) * 5 : for more than 1 probe
        dNTPs 5 µl (N+1) * 5 : pipette 19 µl to the
        Bio/Dig-dUTundefined 2 µl (N+1) * 2 : DNA+H 2 O
        DNase (1:2000) 1 µl (N+1) * 1 :  
        Pol 1 µl (N+1) * 1 :  
        --------------------- --------------------- ---------------------  
        DNA+H 2 O 31 µl    
          =====    
          50 µl    

        Pipette on ice!

        incubation for 2 hrs at 15℃ --> put probes on ice --> test 5 µl of the mix in an agarose electrophoresis, optimal length of DNA fragments should be between 100-1000 bp (in the mean time store probes at -20℃)
        -->if neccessary incubate longer after addition of new DNAse and Pol
        -->add 2.5 µl EDTA (0.5 M, pH 8.0) and 2.5 µl SDS (20%) to stop the reaction, keep the probes at -20℃ until hybridization

        Optimal fragment length after nick translation

        DNA after agarose gel ===> Detection of labeled DNA by a color reaction
        electrophoresis   after transfer to a nylon membrane

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