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 |
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