DNA labeling by nick translation
互联网
			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 | 









