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DNA labeling by nick translation
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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:
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| 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 H2 O |
| DNase (1:2000) | 1 μl | (N 1) * 1 : | ? |
| Pol | 1 μl | (N 1) * 1 : | ? |
| --------------------- | --------------------- | --------------------- | ? |
| DNA H2 O | 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℃ --> 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
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| DNA after agarose gel | ===> | Detection of labeled DNA by a color reaction |
| electrophoresis | ? | after transfer to a nylon membrane |
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