CGH Protocol-DNA labeling by nick translation

Thursday, January 29, 2004

Description
CGH is a molecular cytogenetic method of screening a tumor for genetic changes. The alterations are classified as DNA gains and losses and reveal a characteristic pattern that includes mutations at chromosomal and subchromosomal levels.

Procedure
reagents:
DNA for labeling (concentration c > 150 ng/l)
modified nucleotides: Biotin-16-dUTP, Digoxigenin-11-dUTP, conc. 1nmol/l (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/l (e.g. Boehringer Mannheim)
EDTA (0.5 M, pH 8.0)
SDS (20%)

for one NT reaction 5 g of DNA is used:

Mix (V total = 50 l): 1 probe mix for N probes
NT (10x) 5 l (N+1) * 5 :
b-ME 5 l (N+1) * 5 : for more than 1 probe
dNTPs 5 l (N+1) * 5 : pipette 19 l to the
Bio/Dig-dUTP* 2 l (N+1) * 2 : DNA+H2O
DNase (1:2000) 1 l (N+1) * 1 :
Pol 1 l (N+1) * 1 :
--------------------- --------------------- ---------------------
DNA+H2O 31 l
=====
50 l

*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 15C --> put probes on ice --> test 5 l 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 -20C)
-->if neccessary incubate longer after addition of new DNAse and Pol
-->add 2.5 l EDTA (0.5 M, pH 8.0) and 2.5 l SDS (20%) to stop the reaction, keep the probes at -20C 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

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CGH Protocol-DNA labeling by nick translation