Agilent dna labeling kit

The array CGH was performed using an Agilent Human Genome CGH microarray 180 K kit (Agilent Technologies Inc. Shanghai, China) with a resolution of 6.4 kb. For each sample, 1.5 μg of genomic DNA was used for each array CGH experiment. The array CGH experiments were performed according to the manufacturer’s instruction as described previously
[12 (link)]. Briefly, genomic DNA from patients and controls was digested with the restriction enzymes AluI and RsaI (Promega) and fluorescently labeled with Cy5 (patients) or Cy3 (controls) using the Agilent DNA Labeling Kit. Labeled DNAs were combined, denatured, pre-annealed with Cot- DNA (Invitrogen) and blocking reagent (Agilent), and then hybridized to the arrays for 40 hours in a rotating oven (Agilent Technologies) at 65°C and a speed of 20 rpm. After performing the hybridization step and the recommended washes, the arrays were scanned at 5 μm resolution with an Agilent G2505A scanner. The images were analyzed using the Feature Extraction software (Agilent Technologies, Shanghai, China). All array data that passed the quality metrics were further processed by the DNA Analytics software.

For two-colour microarray, the test immunoprecipitated DNA fragments and 10 ng of input (control) DNA fragments (only digested by BmgT120I, without IP by N45.1 monoclonal antibody) were treated with proteinase K at 37°C for 1 h, followed by phenol–chloroform extraction and ethanol precipitation. The following oligonucleotides were used as adaptor DNA: 5′-GNCTGCGGTGA-3′ and 5′-AGCACTCTCCAGCCTCTCACCGCA-3′; complementary sequences are underlined. Ligations of the adaptor to the test and input DNA were performed using Ligation Pack (Nippon Gene, Toyama, Japan), following the standard protocol. The fragments with the adaptor were amplified with two PCR cycles.
The test and input DNA (800 ng per sample) were labelled with Cy5 and Cy3, respectively, using the standard protocol for Agilent DNA Labeling Kit (Agilent Technologies), and the mixed samples were applied to the Rat Genome CGH Microarray Kit 244A (Agilent Technologies), following the manufacturer's instructions. The arrays were scanned using the Agilent DNA Microarray Scanner G2505B (Agilent Technologies). The scanned data were subjected to image analysis using Agilent's Feature Extraction Software (v10.5.1.1; Agilent Technologies) with the default parameters. Two biologically replicated microarray experiments were performed.