# Microbial Depolymerization of Epoxy Resins: A Novel Approach to a Complex Challenge
## 2 Materials and Methods
### Experimental Set-Up
#### 2.2.4 Genetic Identification of Isolated Microbes  
Fungal and bacterial strains isolated from the microbiological water containing old epoxy samples were identified by DNA sequencing. Two colonies were analyzed separately for each isolate. Bacterial DNA extraction was carried out according to the instruction of the InstaGene method (6% \(w/v\) Chelex resin, BioRad, Italy). To perform the PCR, 1.5 \(\upmu\)L of each extracted DNA sample was added to the PCR reaction mix. The reaction mix was composed of 2.5 \(\upmu\)L QIAGEN Buffer, 1.5 \(\upmu\)L QIAGEN MgCl\({}_{2}\), 0.125 \(\upmu\)L HotStar Taq Polymerase (HotStar Taq Polymerase 1000 Units QIAGEN kit, QIAGEN, Netherlands), 0.5 \(\upmu\)L oligonucleotides Primer UniL (5\({}^{\prime}\)ATTCTAGAGTTTGATCATGGTCA 3\({}^{\prime}\)), 0.5 \(\upmu\)L oligonucleotides Primer UniR (5\({}^{\prime}\)ATGGTACCGTGTGACGGCGGTGTGA 3\({}^{\prime}\)), 0.5 \(\upmu\)L PCR Nucleotide Mix Plus (dUTP/dATP/dGTP/dCTP) and 17.875 \(\upmu\)L H\({}_{2}\)O [25; 26]. After the quantification of DNA with the Nanodrop spectrophotometer (ThermoFisher Scientific), 1.5 \(\upmu\)L of purified DNA was added to the reaction mix containing 2 \(\upmu\)L of primer (UniL for one mix, UniR for the other), 1 \(\upmu\)L of BigDye(r) Terminator v3.1 Ready Reaction Mix (ThermoFisher Scientific) containing the marked dNTP, 5 \(\upmu\)L of 5 for BigDye(r) Buffer 5 for (ThermoFisher Scientific) and 4 \(\upmu\)L of H\({}_{2}\)O for a total of 10 \(\upmu\)L of volume. The amplification was performed in the Veriti AB Prism thermocyler with the following routine: 96 \({}^{\circ}\)C for 1 min, 25 repetitions of 96 \({}^{\circ}\)C for 10 s, 50 \({}^{\circ}\)C for 5 s, 60 \({}^{\circ}\)C for 4 min and then cooling down and maintaining at 4 \({}^{\circ}\)C. Lately, the PCR products were filtrated by centrifugation using a column containing the Sephadex G-50 matrix (10 g of Sephadex G-50 matrix in 150 mL milliQ H\({}_{2}\)O) in order to retain the non-incorporated fluorescent nucleotide and recover the PCR products. Some columns (tips cut just below the filters level) were filled with 900 \(\upmu\)L of a suspension of Sephadex G-50 and placed in QIAGEN collection tubes. The tubes containing the columns and the suspension were centrifuged for 3 min at 2700 rpm in order to solidify the matrix. The columns containing the solid matrix were recovered from the collection tubes and transferred in two 1.5 mL Eppendorf tubes. Measures of 10 mL of each of the BigDye reaction mixtures were transferred in the two columns and centrifuged for 3 min at 2700 rpm. The columns containing the matrix were removed, and 5 mL of HiDi Formamide (ThermoFisher Scientific) were added to each sample. Measures of 15 mL of the purified DNA and HiDi Formamide from each sample were transferred in two wells of a microtiter plate. The microtiter plate containing the amplified 16 s DNA was then loaded in the sequencer (Applied Biosystems 3500 Genetic Analyzer, Thermo Fisher Scientific) and the sequencing reaction was performed. The presence of incorporated fluorescent dNTP allows the identification of the nucleotide composition of the sequenced fragments. Concerning fungi, fungal cultures were inoculated in the isolation medium and covered with a cellulose film in order to remove the mycelium from the solid medium. A measure of 508 mL of sterile TE buffer (1.211 g/1 Trizma base, Sigma; 0.372 g Na\({}_{2}\)EDTA\({}_{2}\)H\({}_{2}\)O, Fluka) was added in an Eppendorf tube together with 1 cm\({}^{2}\) of fungal culture. Then, 50 mL lysozyme (10 mg/mL Lysozyme, Sigma) was also added, and the sample was incubated 2 h at 37 \({}^{\circ}\)C. After that, 30 mL SDS 10% (Sigma) and 12 mL proteinase K (8.3 mg/mL, Sigma) were added to the sample that was incubated a second time for 24 h at 37 \({}^{\circ}\)C, in order to complete the breakdown of the cell walls. After the second incubation, 100 mL NaCl 5M (Sigma) and 80 mL sterile CTAB 10% (10 g CTAB, Merck; 100 mL NaCl 0.7M, Sigma) were added and the sample was heated at 65 \({}^{\circ}\)C for 10 min.