Dig high prime dna labelling kit

High-throughput specificity assays were carried out using a dot blot platform, essentially as previously described (Albuquerque et al. 2011 (link)). PCR amplicons obtained using primers Psm1-6F/6R, with template DNA from strain Psm 28a (race 1), and primers Psm2-8F/8R, with Psm 77 (race 2), were purified using the GFX PCR and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) and labelled with digoxigenin, using the DIG-High Prime DNA labelling kit (Roche, Basel, Switzerland) in order to obtain the two tested hybridisation probes Psm1 and Psm2, respectively.
Amounts of 100 ng of heat-denatured DNA from each bacterial strain were transferred to a nylon membrane using a Bio-Dot apparatus (Bio-Rad, Hercules, USA). Hybridisation was carried out overnight at 68 °C with a final probe concentration of 100 ng/mL, and the washing and detection steps were carried out according to the DIG application manual (Roche). The chemiluminescent signal indicative of probe–target hybrids was detected using a Molecular Imager ChemiDoc XRS+ System (Bio-Rad), with all pixels below saturation point.

Genomic DNA (20 µg) isolated from WT V592 or the indicated mutant strains was digested with the indicated restriction enzymes. Digested DNA was separated by electrophoresis on an agarose gel overnight and transferred onto a nylon membrane. Gene‐specific probes were labelled using the DIG High Prime DNA Labelling Kit and the presence of corresponding DNA fragments was detected by the Detection Starter Kit I (Roche, Indianapolis, IN, USA).