This is a cached page for the URL ( To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
DNA labeling by nick translation

DNA labeling by nick translation

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 MgCl2, 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-dUTP* 2 µl (N+1) * 2 : DNA+H2O
DNase (1:2000) 1 µl (N+1) * 1 :
Pol 1 µl (N+1) * 1 :
--------------------- --------------------- ---------------------
DNA+H2O 31 µl
50 µl

*in the standard protocol Tumor DNA is labeled with Bio-dUTP, Normal DNA is labeled with Dig-dUTP

Pipette on ice!

incubation for 2 hrs at 15°C --> 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°C)
-->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°C 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

nick translation