Dna clean concentrator 25 kit

Nucleotide sequences of V. aucta mitochondrial partial cytochrome oxidase subunit 1 (COI) gene (accession: FM178086.1) and nuclear partial non-coding internal transcribed spacer 2 (ITS2, accession: FM178155.1) were retrieved from the NCBI GenBank [27 (link)]. These regions were chosen because they are species-specific within Verrallia species according to Kehlmaier and Assmann [28 (link)]. Sequences were submitted to Primer3 software [29 (link)] and one primer pair was selected for each target (Table 2). Primers were tested in conventional PCR on DNA extracted from the larvae of V. aucta isolated from parasitized adults of P. spumarius and N. campestris, and in order to verify their specificity, on DNA extracted from non-parasitized adults of P. spumarius. PCR products were purified with the DNA Clean & Concentrator-25 kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s instructions and send to BMR Genomics (Padova, Italy) for sequencing.

LlaGI was purified as previously described (18 (link)). A modified version of pRMA03 (18 (link)) was used as a substrate. pRMA03 was digested using SmaI and PshAI and re-ligated generating pRMA03S, a substrate in which the two LlaGI sites are in head-to-head orientation separated by 997 bp. A reaction mix containing 2 nM pRMA03S, 200 nM LlaGI in TMD Buffer (50 mM Tris–Cl, pH 8.0, 10 mM MgCl₂, 1 mM DTT) was pre-incubated at 25°C for 5 min. Cleavage reactions were initiated by the addition of 4 mM ATP, and stopped at 10, 30 or 60 s by adding binding buffer from the DNA Clean & Concentrator-25 kit (Zymo Research) supplemented with 30 mM EDTA and 37 mM sodium acetate. Purification of cleaved DNA proceeded as described by the manufacturer.

Extraction kits were compared by quantification cycle (C_q) values, with lower C_q values indicating more-efficient amplification, which, in this design, we interpreted as an effect of the extraction method. Mean values were calculated based on triplicate runs after confirmation of amplicon size by melting curve analysis. All statistical tests were performed by using JMP Pro version 12.0.1 (SAS Institute, Cary, NC, USA). Statistically significant differences between tissue values were determined by a Kruskal-Wallis test at an alpha value of 0.05. For blood, Kruskal-Wallis statistics included a blocking factor of replicate number. The differences between unconcentrated and concentrated DNAs by the Zymo Research DNA Clean & Concentrator-25 kit were compared by a Wilcoxon signed-rank test. Statistical significance was determined as a P value of <0.05.

Methylation Primer Express Software V1.0 was used to design bisulfite sequencing PCR (BSP) primers, which are provided in Table S1. DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, CA, USA) according to the manufacturer’s protocol. We acquired bisulfite converted DNA using the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocols. PCR was carried out with the ZymoTaq PreMix (Zymo Research, CA, USA) according to the manufacturer’s specifications. The PCR products were subsequently purified using the DNA Clean & Concentrator - 25 Kit (Zymo Research). Subsequently, the PCR products were cloned into a TA vector (Invitrogen, Carlsbad, CA, USA). Ten effective subclones were selected for each gene, and successful cloning was subsequently confirmed by analyzing the sequence data (BiQ Analyzer V2.0) obtained with an ABI 3730 DNA sequencer (Applied Biosystems, Foster City, CA)29 (link).

Genomic DNA was extracted from three males and three females of C. capitata Benakeion, SEIB, VIENNA 8, wp tsl, and D53 tsl strains using ExtractMe DNA tissue kit (Blirt, Poland) following the manufacturer’s instructions. A NanoDrop spectrometer was used to assess the quantity and quality of the extracted DNA. Primers (Table S2) were designed using the Geneious Prime 2022.1.1 software. PCRs were performed in a 25 µL reaction volume using 12.5 μL Platinum™ Green Hot Start PCR Master Mix (2X) Kit (Thermo Fisher Scientific), 60–80 ng DNA, and the following PCR settings [94 °C, 2 min; 35 cycles of (94 °C, 30 s; 60 °C, 30 s; 72 °C, 120 s); 72 °C, 5 min]. PCR products were analyzed by electrophoresis in 2% agarose gels and visualized under UV light. Amplicons were purified using the DNA Clean & Concentrator-25 kit according to the manufacturer's protocol (Zymo Research—Irvine, CA, USA). The purified products were adjusted to the concentration of 10 ng/µl while sequencing primers were diluted following the Eurofins Genomics instructions up to 100 nmol/µl. The sequencing mix was prepared in a final volume of 15 µl (13 µl of DNA and 2 µl of primer). Sequencing results were imported in Geneious Prime 2022.1.1 and aligned to the Ccdor gene wild-type sequence extracted from Ccap 3.2.1 (accession GCA_905071925.1) using the Geneious Prime “Map to reference” tool with default parameters.

A chloramphenicol resistance cassette flanked by Hpy188I restriction sites was amplified by overhanging-end PCR from the pACYC184 plasmid (19 (link)) using oligos 5′-CTAGCTTCAGAAGTCAGCGACTCGCATCACGCACCAATAACTGCCTTAAAAAAATTACGC-3′ and 5′-TACGTATCAGAGACGTAGCGTACGCATCGTCAGCGAAAATGAGACGTTGATCGGC-3′. Template plasmid was eliminated by treating with DpnI for 1 h at 37°C. The PCR product was purified using DNA Clean & Concentrator-25 kit (Zymo Research). Purified PCR product was digested with Hpy188I (New England Biolabs) for 2 h at 37°C to generate single dT 3′ overhangs, and purified.