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℃)
--
--
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
