Mj mini gradient thermal cycler

The extraction of the bacterial genomic DNA from the selected isolate
was performed using Genomic Mini kit (A&A biotechnology, Poland).
The cell pellets were incubated with lysozyme at 37 °C for 20
min. By using an MJ Mini Gradient Thermal Cycler, polymerase chain
reaction (PCR) was performed (Bio-Rad, Hercules, CA, USA). The universal
primers 27F and 1492R (5′-AGAGTTTGATCCTGGCTCAG-3′/5′-GGTTACCTTGTTACGACTT-3′)
were employed to amplify the 16S rRNA gene. The PCR reaction was carried
out according to our previous report.^{19 (link)} The 16S rRNA gene’s nucleotide sequences were edited, put
together, and aligned. Moreover, the MEGA 11 sequence alignment software
(version 11.0.11) was used to generate consensus sequences, which
were then tested using the BLAST program. The 16S rRNA gene’s
nucleotide sequences were utilized to establish genetic connections
using the neighbor-joining method.^{19 (link)}

All mold isolates were characterized based on the macroscopic and microscopic characteristics of their colonies. They were then grouped into strains and identified using taxonomic keys after culturing them on MEA and Czapek–Dox Agar (Difco, Detroit, MI, USA) media, [24 ,25 ,26 ,27 ,28 (link)].
Genomic DNAs of all mold strains were extracted using the FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH, USA) following the manufacturer’s protocol, except for some modifications to the first step: samples were homogenized twice for 1 min, with one intervening minute on ice, instead of only once for 40 s. PCR was performed using an MJ Mini Gradient Thermal Cycler (Bio-Rad, Hercules, CA, USA). Universal primers ITS1 and ITS4 were used for the amplification of the internal transcribed spacer regions (ITS1/ITS2) [29 ]. The amplification of actin and calmodulin gene fragments was performed with primers Act-for, Act-rev and Calm-for, Calm-rev described by Lawrence et al. [30 (link)]. Primer sequences used in this study are presented in Table 2. Each PCR reaction was carried out in 50 µL volume containing 40 pmol of each primer, 1.5 U of RedTaq ReadyMix DNA polymerase (Sigma-Aldrich, St. Louis, MO, USA), 20 ng of template DNA and made up to 50 µL with PCR grade water. PCR products were detected by 1% (w/v) agarose gel electrophoresis in 0.5 × TBE buffer (Sigma-Aldrich).

Total RNA from the rat liver was extracted using the TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. The purity and concentration of RNA were determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). cDNAs were synthesized from 1 μg of total RNA using a PrimeScript 1st strand cDNA Synthesis Kit (TaKaRa, Dalian, China) according to the manufacturer's recommended protocol. PCR was performed with 2x Es Taq MasterMix (CWBIO, Beijing, China) in an MJ Mini Gradient Thermal Cycler (Bio-Rad) using the following parameters: 94°C denaturation for 3 min followed by 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s and a final extension at 72°C for 3 min. GAPDH was amplified from the same cDNA samples as an internal control. The amplified PCR products were analyzed in 2% agarose gels, and a semiquantitative analysis of the band intensities was performed with Gene Tools software (UVP, Inc., Upland, CA, USA). The intensities of the bands were normalized against that of GAPDH. The following primer sequences for RT-PCR were used: GK F, 5′-TCAACTACAGAAAATGGCGGAA-3′, and R, 5′-CCAGAACTGTAAGCCACTCG-3′; GAPDH F, 5′-ATTCAACGGCACAGTCAA-3′, and R, 5′-CTTCTGGGTGGCAGTGAT-3′.

Total RNA was purified from limb tissues or blood samples using Nucleospin kit II (740955.50; Macherey-Nagel GmbH & Co. KG, Düren, Germany), and cDNA was synthesized using Superscript II reverse transcriptase (18064-014; Invitrogen in Thermo Fisher Scientific, Tokyo, Japan) with oligo(dt) 12–18 primers (18418012, Invitrogen in Thermo Fisher Scientific). In the case of limb tissues (Fig. 1g), the mass of blastema samples was weighed and an equivalent mass of intact limb samples was harvested (see Fig. 1a), and these samples were processed for RNA purification and cDNA synthesis at the same scale. Genomic DNA was purified from blood samples using the Wizard Genomic DNA Purification Kit (A1120; Promega, Madison, WI, USA). Using these DNA samples, PCR was carried out with the KODFX system (KFX-101, Toyobo, Osaka, Japan) on an MJ Mini Gradient Thermal Cycler (PTC-1148; Bio-Rad, Hercules, CA, USA). Primer sets used in this study and cycle numbers for the data in figures are listed in Supplementary Table S4. PCR products were subcloned into Escherichia coli using a TA cloning system (45-0640, TOPO TA Cloning Kit, Dual Promoter; Thermo Fisher Scientific) and then sequenced by Sanger protocols (ABI 3130; Applied Biosystems, in Thermo Fisher Scientific)^{26 (link)}.

2-2-Diphenyl-1-picrylhydrazyl (DPPH); Folin-Ciocalteu’s reagent (2 N); (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox); (+)-catechin; and 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), linoleic acid, Tween 40, iron (II) sulfate heptahydrate, iron (III) chloride hexahydrate, β-carotene, RedTaq ReadyMix DNA polymerase, agarose gel electrophoresis and TBE buffer were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Plate count agar (PCA), DG18 medium, and bullion agar were purchased from Merck (Darmstadt, Germany). The oxidase test kit was obtained from Merck (Darmstadt, Germany). API Tests were purchased from BioMérieux (France). Genomic Mini kit and Clean Up Mini Kit were purchased from A&A Biotechnology (Gdynia, Poland). MJ Mini Gradient Thermal Cycler was obtained from Bio-Rad, (Hercules, CA, USA). BigDye Terminator Ready Reaction Cycle Sequencing kit was purchased from Applied Biosystems (Foster City, CA, USA). Unless indicated otherwise, all chemicals were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland).

PCR amplification was performed using a total reaction volume of 25 μL which contained 20 ng of DNA, buffer (67 mM Tris-HCl, pH 8.8, 16 mM (NH₄)2SO₄, 0.01% Tween 20), 3 mM of MgCl₂, 1.0 U of SuperHot Taq DNA Polymerase (Genaxxon Bioscience GmbH, Ulm, Germany), 280 nM of each primer and 200 μM of dNTP (Grisp, Porto, Portugal) (Table 2). The reactions were carried out in a MJ Mini™ Gradient Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA), using the following optimized programs: initial denaturation at 95 °C for 5 min; 35 or 40 cycles (for EG-F/EG-R or Gkb2-F/Gkb2-R primers, respectively) of amplification at 95 °C for 30 s, 63 °C or 62 °C (for EG-F/EG-R or Gkb2-F/Gkb2-R primers, respectively) for 30 s and extension at 72 °C for 30 s; and a final extension at 72 °C for 5 min.
PCR products were further analyzed by electrophoresis in a 1.5% agarose gel stained with 1× Gel Red (Biotium, Hayward, CA, USA) and running in 1× SGTB buffer (GRISP, Porto, Portugal) for 20–25 min at 200 V. Each extract was amplified in at least two independent assays.