Digoxigenin 11 dutp dig dutp

ChIP clones with insert DNA fragments confirmed by colony PCR were further checked for genomic abundance by dot blot analysis. The PCR product (2 μL) of each clone was dotted onto a positively charged nylon transfer membrane (Amersham Hybond-N+, Global Life Sciences Solutions USA, Marlborough, MA, USA) and cross-linked by UV under 0.120 J/cm² in a Bio-Link Crosslinker BLX-254 (Witec AG, Sursee, Switzerland). Simultaneously, the plasmid DNA of Lycoris 5S rDNA (La5S rDNA: GenBank accession number MW036652) was used as a control. Genomic DNA of L. aurea was boiled for 30 min to obtain DNA fragments < 10 kb before being labeled with Digoxigenin-11-dUTP (Dig-dUTP; Roche, Germany) by using Nick Translation Mix (Roche, Germany). The membrane was incubated with Dig-labeled genomic DNA probes in hybridization buffer containing 5X Saline Sodium Citrate buffer (SSC), 5X Denhardt solution and 0.5% SDS. After hybridization overnight at 37°C, the membrane was washed with TBST buffer (20 mM Tris-HCl pH 7.6, 200 mM NaCl, and 0.1% Tween 20) and immune-detected with anti-Dioxigenin antibody (in 1:2500 dilution, Abcam, UK) as the primary antibody and anti-mouse IgG conjugate HRP antibody (1:5000 dilution, Abcam, UK) as the secondary antibody.

Cells were labeled with 100 µM EdU (Life Technologies, Carlsbad, CA, USA) for 10 min, aliquoted onto coverslips and fixed with 4% paraformaldehyde in PBS, and then permeabilized with 0.5% Triton X-100 in PBS. Blocking was performed by incubation in 3% BSA/0.1% Tween 20/Tris-buffered saline, pH for 30 min. EdU-labeled DNA was detected with the Click-iT EdU Alexa Fluor 488 Imaging Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Digoxigenin (DIG)-labeled DNA was detected with an anti-DIG antibody conjugated with FITC (1:200 dilution; Roche, Mannheim, Germany). DAPI was used to counterstain the DNA. In the experiment in Figure 1B, digoxigenin-11-dUTP (DIG-dUTP; Roche, Mannheim, Germany) was loaded into cells with the hypotonic shift method for replication labeling [37 (link)]. Incorporated DIG-dUTP was detected with an anti-DIG antibody conjugated with rhodamine (Cat#11207750910; Roche, Mannheim, Germany).