DNA labeling by nick translation

 

DNA labeling by nick translation

reagents:
DNA for labeling (concentration c > 150 ng/µl)
modified nucleotides: Bio tin-16-dUTP, Dig oxigenin-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 MgCl 2 , 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-dUTundefined	2 µl	(N+1) * 2 :	DNA+H 2 O
DNase (1:2000)	1 µl	(N+1) * 1 :
Pol	1 µl	(N+1) * 1 :
---------------------	---------------------	---------------------
DNA+H 2 O	31 µl
	=====
	50 µl

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