Dig dna labeling mixture

The metaphase chromosome spread was prepared according to a standard protocol, which involved hypotonic swelling in 75 mM KCl, at 37 °C for 10 min, the centrifugal washing by methanol/acetic acids = 3/1 for three times, followed by dropping onto the prewet slide glass (superfrost, Matsunami Glass Ind. Ltd). The digoxigenin (DIG)-labeled probe for detecting the amplified plasmid sequence was prepared from pΔBM d2EGFP plasmid DNA using the BioPrime DNA labeling system (Invitrogen) combined with 10× DIG DNA labeling mixture (Roche Diagnostics). The hybridized DIG probe was detected with an anti-DIG fluorescein Fab fragment (Roche Diagnostics). The slide was counterstained with 4′,6-diamidino-2-phenylindole and examined using an epifluorescence microscope (TE2000E, Nikon) equipped with a 100× objective lens (Nikon Plan Fluor, NA 1.30 oil) and an appropriate filter set. Digital images were acquired with a Nikon D7000 digital camera and processed with Adobe Photoshop CS6 (Adobe Systems, Inc). For flow cytometry analysis of d2EGFP expression, the cells were resuspended in phosphate-buffered saline and analyzed using a FACSCalibur system (Becton Dickinson Co) in the absence or the presence of 2 mM sodium butyrate for the last 3 days of culture.

Plasmid pSFVdhfr was used to prepare a probe for detection of the plasmid sequence in clone 12 (DM) and clone 22 (HSR) cells [5 (link)]. Cosmid DNA (c-myc) was used to detect natural amplicons in COLO 320DM and COLO 320HSR cells [6 (link)]. Probes were DIG- or biotin-labeled using the BioPrime DNA Labeling System (Invitrogen) with or without 10×DIG DNA Labeling Mixture (Roche Lifescience Inc.). The probe was hybridized to metaphase chromosome spreads, and hybridized DIG- and biotin-labeled probes were detected using anti-Digoxigenin-Fluorescein Fab fragments (Roche) or Streptavidin Alexa Fluor 594 conjugate (Invitrogen), respectively.

Metaphase chromosome spreads were prepared by a standard protocol [21 ]. Chromatin fibers were prepared according to our previously published protocol [5 (link)], which was developed based on the preceding protocol [22 (link)]. DIG-labeled plasmid probes were prepared from pG5 or pKV-AR1 plasmid DNA using the BioPrime DNA Labeling Kit (Invitrogen) and 10× DIG DNA Labeling Mixture (Roche). Biotin-labeled G5 probe was prepared by PCR amplification of the G5 sequence (966 bp) in the pG5 plasmid using the primer set used for the preparation of repeat DNA (see above) and biotin labeling of the product using the Biotin PCR labeling kit (Promokine). The probe was hybridized to metaphase chromosome spreads, and hybridized DIG- and biotin-labeled probes were detected using anti-Digoxigenin-Fluorescein Fab fragments (Roche) or Streptavidin Alexa Fluor 594 conjugate (Invitrogen), respectively. For the flow cytometric analysis to evaluate d2EGFP expression, the cells were resuspended in phosphate buffered saline and analyzed using FACS Calibur (Becton Dickinson Co.).