2.10. Colony PCR for Bacterial Identification after Nanofiber Dissolution

Colony PCR was performed to confirm the identity of bacterial colonies grown on solidified MRS media after their release from nanofibers. Primers (Integrated DNA Technologies) were designed to amplify a short genomic DNA fragment (200–400 base pairs) identified using IMG/M (https://img.jgi.doe.gov/m/ (accessed on 23 May 2022)) and were specific for each of the species tested (Table 1). Bacterial colonies were transferred to a PCR mixture (DreamTag^TM DNA Polymerase, Thermo Scientific, Waltham, MA, USA). The samples were heated at 99 °C for 10 min to lyse the bacteria, followed by the addition of Tag DNA Polymerase (Thermo Scientific). The PCR conditions were as follows: denaturation at 94 °C for 30 s, annealing at 56 °C for 1 min, and extension at 72 °C for 1 min for 30 cycles, followed by a 5 min extension at 72 °C. After the PCR reaction, the samples were loaded in 1.5% agarose (Sigma Aldrich, Darmstadt, Germany) gel and visualized under UV light. Positive control from frozen stocks and negative control without bacteria were also included.

Species specific primers for colony PCR.