DNAlabelingbynicktranslation

DNA labeling by nick translation

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 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:

*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