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:
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