# Biodeterioration of Salon de Reinos, Museo Nacional del Prado, Madrid, Spain
## 2 Materials and Methods
### DNA Extraction and Amplification  
The extraction of DNA from the strains was carried out from bacterial biomass and fungal mycelia. The biomass was transferred to a 1.5 mL Eppendorf tube containing 500 \(\mu\)L of the TNE extraction buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA; pH 8) and 2 and 4 mm glass beads. The mixture was homogenized using a stirrer (Fast Prep-24), applying two cycles of 45 s at a speed of 4.5 m/s. The proteins were removed by adding 50 \(\mu\)L of sodium dodecyl sulfate (10% \(w/v\)) and 70 \(\mu\)L of proteinase K (10 mg/mL), and the mixture was incubated at 50 \({}^{\circ}\)C for one hour. DNA was purified with 750 \(\mu\)L phenol-chloroform-isoamyl alcohol (25:24:1). The mixture was centrifuged at 8000 rpm for 5 min. The supernatant obtained was subjected to further purification with 450 \(\mu\)L of chloroform-isoamyl alcohol (24:1). Centrifugation was repeated under identical conditions, and the supernatant was collected. DNA precipitation was performed by adding 50 \(\mu\)L of 3 M sodium acetate (pH 5.2) and 1 mL of absolute ethanol. To promote precipitation, the mixture was incubated at \(-\)20 \({}^{\circ}\)C for 30 min and centrifuged at 13,000 rpm for 10 min. The precipitate was washed with ethanol (70%) and allowed to dry at room temperature. Finally, the precipitated DNA was then dissolved in 100 \(\mu\)L of sterile ultra-pure water. The resulting DNA concentration in the extraction products was quantified using a Qubit 2.0 fluorimeter (Invitrogen), following the manufacturer's instructions. The products were stored at \(-\)80 \({}^{\circ}\)C until use. For the identification of bacteria, the 16S rRNA gene was amplified, and for fungi, the ITS region was between the small and large subunit of ribosomal RNA, 18S rRNA and 28S rRNA (ITS1 and ITS2, respectively). The primers 616F (5\({}^{\prime}\)-AGA GTT TGA TYM TGG CTC AG-3\({}^{\prime}\)) [17] and 1522R (5\({}^{\prime}\)-AAG GAG GTG ATC CAG CCG CA-3\({}^{\prime}\)) [18] were used for \begin{table}
\begin{tabular}{c c c} \hline \hline
**Sample** & **Description** & **Location** \\ \hline M1 & Surface dust & S-3. Mexico shield \\ M2 & Black stains & S-4. Burgundy shield \\ M3 & Black stains & S-02. Between Galicia and Murcia shields \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Description and location of the samples studied. bacteria, and the primers ITS1 (5\({}^{\prime}\)-TCC GTA GGT CCT GAA GGG G-3\({}^{\prime}\)) and ITS4 (5\({}^{\prime}\)-TCC TCC GCT TAT TGA TAT GC-3\({}^{\prime}\)) described by [PERSON] et al. [19] were used for fungi. Each PCR reaction was run in a final volume of 50 \(\upmu\)L. The reaction mixture consisted of 5 \(\upmu\)L of 10X reaction buffer [750 mM Tris-HCl (pH 9), 500 mM KCl, 200 mM (NH4)2 SO4], 2 \(\upmu\)L of 50 mM MgCl2, 0.5 \(\upmu\)L of each 50 \(\upmu\)M primer (Invitrogen Life Technologies), 5 \(\upmu\)L of deoxyribonucleotide mix (dNTPs) (Bioline GC Biotech, Alphen aan den Rijn, The Netherlands), 2 mM each, 0.25 \(\upmu\)L of Taq polymerase enzyme (5 U/\(\upmu\)L) (Biotools M&B Labs, Madrid, Spain), and 10-20 ng of template DNA; the reaction volume was completed with sterile ultra-pure water. All samples were run in duplicate, and two negative controls, containing sterile ultra-pure water replacing template DNA, were included for each batch of reactions. The PCR reactions were performed in an iCycler thermal cycler (Bio-Rad, CA, USA).