Ready to go pcr beads

DNA was extracted from apices of stems and branches from dried herbarium specimens using the DNeasy Plant Mini Kit for DNA isolation (Qiagen). We selected four loci previously used for phylogenetic reconstructions of Orthotrichum [33 ,45 (link)]: three chloroplast loci, namely atpB-rbcL, rps4, and trnL-F, and the nuclear internal transcribed spacer II (ITS2). The primer pairs used for each locus were atb1/rbcL1 [50 ], rpsA/trnaS [51 ,52 (link)], trnC/trnF [53 (link)] and ITS2F/ITS2R [54 ].
Double-stranded DNA templates were prepared by PCR, which was performed using Ready-To-Go PCR Beads (Amersham Pharmacia Biotech Inc.) in a final reaction volume of 25 μL according to the manufacturer’s instructions. PCR amplification of atpB-rbcL, rps4, and trnL-F was performed using the protocol described in [33 ], while the ITS2 protocol followed [45 (link)]. PCR products were purified using the Exo/SAP protocol (Thermo Fisher Scientific, Spain). Samples were incubated with 1 μL of Exo1 enzyme and 4 μL of FastAP following the manufacturer’s instructions. Cleaned PCR products were sequenced by Macrogen (www.macrogen.com). All new sequences were deposited in GenBank (see S2 Appendix).

Parasitoid DNA was extracted from single ethanol‐preserved specimens following the protocol from the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA). A 658‐bp fragment from the 5′ region of MT‐CO1 was amplified using the LCO (light DNA strand CO1) and HCO (heavy DNA strand CO1) primers (Folmer et al., 1994) using Ready‐To‐Go PCR beads (Amersham Pharmacia Biotech, Amersham, UK) on the following program: 5 min 94 °C hotstart; 40 cycles: denature 94 °C for 15 s, anneal 46 °C for 15 s, extend 72 °C for 15 s; final extension 72 °C for 10 min. This gene has been used in previous studies of braconid phylogenetics (Belshaw et al., 2000; Belshaw & Quicke, 2002; Dowton et al., 2002; Zaldivar‐Riverón et al., 2006; Sharanowski et al., 2011; Stigenberg & Ronquist, 2011). Product yield and specificity were observed using agarose gel electrophoresis. PCR products were purified with EXO1 and FastAP. The product was sequenced using both the forward and reverse primers and sequencing reactions were purified with the DyeEx 96 kit (Qiagen). Sequences were assembled and edited using Geneious Pro v.9.1.8. The Voseq v.1.7.3 (Peña & Malm, 2012) database was used for storing voucher and DNA sequence data. BLAST analysis was performed to identify similar sequences within the NCBI database.

Zebrafish RNA was isolated at specific developmental stages (4 h, 10 h, 24 h, 48 h, 72 h, 3.5 d, 4.5 d, and 6.5 d) and used to generate cDNAs using Retroscript (Ambion, Austin, TX, USA). cDNA was used as a template in PCR reactions with primers flanking the variable region of the Col11a1a chain. Ten picomoles of each primer and 2 µL of cDNA were added to 22 µL of PCR master mix generated by adding water to Ready-To-Go PCR Beads (Amersham Biosciences, Piscataway, NJ, USA). The final reaction (25 µL) contained 1.5 U Taq DNA Polymerase, 10 mM Tris-HCl, pH 9.0 at room temperature, 50 mM KCl, 1.5 mM MgCl₂, 200 µM of each dNTP as well as bovine serum albumin (BSA). Each reaction was then incubated as follows: 95 °C for 5 min, (95 °C for 1 min, 55.5 °C for 1 min, 72 °C for 1 min) × 30 cycles, and 72 °C for 10 min. PCR products were separated by size by electrophoresis on a 2% agarose gel (Nusieve 3:1) in Tris Acetate EDTA (TAE) buffer and stained with ethidium bromide. Bands were visualized using a Kodak ID Image Station (Eastman Kodak Company, Rochester, NY, USA) trans-illuminator.

The primer pairs whose sequences span the site of the transposable intron in the 25S rDNA were those described by McCullough et al. [31 (link)]: CA-NT-L: 5'-ATAAGGGAAGTCGGCAAAATAGATCCGTAA-3' and CA-NT-R: 5'-CCTTGGCTGTGGTTTCGCTAGATAGTAGAT-3'. Amplification reactions were performed in 50 μL of distilled water containing 2.0 μL of each primer, 2.0 μL of genomic DNA (5 μg/mL) and one PCR bead (Ready-to-Go PCR beads; Amersham Pharmacia Biotech, Piscataway, NJ, USA). The PCR conditions used were as follows: denaturation by incubation for 5 min at 93 °C prior to 40 cycles of 93 °C for 30 s, 55 °C for 45 s, and 72 °C for 45 s and a final extension at 72 °C for 10 min. All reaction products were characterized by electrophoresis on 1.5% agarose gels in TBE (Tris-borate-EDTA) buffer at 70 V for 30 min and were then stained in a solution of 0.5 μg of ethidium bromide per mL.
According to the results of electrophoresis, the genotypes of C. albicans can be divided into 5 groups by the size of DNA amplified (450 bp for group A, 840 bp for group B, 450 and 840 bp for group C, 1080 bp for group D and 1400 bq for group E) [32 (link),33 (link)].

The bacteria examined in this work were 100 Nocardia strains recovered from clinical samples (84 of respiratory origin, 6 cutaneous, 2 from the central nervous system and 8 from other tissues) sent to our reference centre (Spanish National Centre for Microbiology) from different hospitals between 2006 and 2014. These were identified by 16S rRNA analysis (see below) as representing 30 Nocardia species, and were selected for the present study since they represented more and less commonly encountered members of the genus. All these bacteria were incubated on buffered charcoal yeast extract agar (BCYE) or Columbia 5% sheep blood agar at 37°C for at least 48 h (i.e., until growth was clearly visible). DNA was extracted by the boiling method and amplifications of the studied genes were performed using Ready-To-Go PCR Beads (Amersham Biosciences, Buchinghamshire, UK). The products were electrophoresed and purified using Exo SAP-ITTM reagent (GE Healthcare, NJ, USA), and sequenced by capillary electrophoresis in a ABI PRISM 3100 apparatus (Applied Biosystems, Foster City, CA, USA) (Carrasco et al., 2013 (link)) PCR primers and conditions are listed in Supplementary Table S1.

Only the tip of a single gametophyte shoot from each sample was selected for DNA extraction to prevent contamination. The rest of the gametophyte, and the sporophyte if present, were preserved in a microscope slide fixed with glycerogelatin to allow identification revision. DNA was extracted using the standard DNeasy Plant Mini Kit protocol (QIAGEN). Nucleotide sequences were amplified by PCR from four genomic regions (Table 4): one of them from to the nuclear genome (ITS2) and the other three from to the plastid genome (rps4, trnG and trnL-F). PCRs were performed using Ready-To-Go™ PCR Beads (Amersham Pharmacia Biotech Inc.) in a final volume of 25 μL, initially with 2 μL of DNA, and in the case of subsequent PCR failure, with up to 10 μL. Amplification protocols are specified in Table 5. Amplification’s success was verified by electrophoresis and PCR products were purified using Exol/FastAP (Thermo Fisher Scientific, Spain) with 1 μL of Exonuclease and 4 μL of FastAP enzymes per tube, applying 37 °C and 85 °C for 15 min each. Finally, cleaned PCR products were sequenced by Macrogen. Two reads were obtained for each product, which were aligned using Geneious 2022.0.2.