DNA实验

DNA labeling by nick translation

2024-10-09 DNA实验加入收藏

reagents: DNA for labeling (concentration c > 150 ng/µl) modified nucleotides

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 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-dUTP*	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

*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