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℃ --> 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
DNA after agarose gel |
===> |
Detection of labeled DNA by a color reaction |
electrophoresis |
|
after transfer to a nylon membrane |
|