Exosap it express pcr product cleanup

F1 juveniles were taken from Petri dishes and digested by proteinase (Thermo Fisher Science) to obtain genomic DNA as described previously (Ohta et al. 2010 (link)). Oral siphon and sperm were surgically obtained from mature animals, and processed for genomic DNA extraction using the QIAamp DNA Micro Kit (Qiagen), or digested with proteinase. The genomic DNA was used for PCR amplification (35 cycles of 95° 30’’, 58° 30’’, 72° 1’) of target regions with Ex Taq HS DNA polymerase (Takara Bio). The PCR products were purified enzymatically with ExoSAP-IT Express PCR product cleanup (Thermo Fisher Scientific), or by NucleoSpin Gel and PCR clean-up (Macherey-Nagel). PCR products were sequenced by Genewiz. The primers used in this study are summarized in Supplemental Table S1.

Ten dried swabs per sampling point for each flock were pooled and immersed in 1 mL of phosphate-buffered saline (Life Technologies Limited, Paisley, UK). RNA was extracted from 140 µL of eluate using QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. Samples were processed using a nested RT-PCR targeting the G gene, which enables the simultaneous detection and differentiation of A and B aMPV subtypes [13 (link)]. When the assay gave a negative result, the nested RT-PCR was repeated on RNA extracted from the remaining 10 swabs from the same sampling point. The obtained G gene amplicons were purified using ExoSAP-IT™ Express PCR Product Cleanup (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions and sequenced in both directions by a commercial sequencing service (Macrogen Europe, Amsterdam, The Netherlands), using the nested RT-PCR primer pair G5- (5′- CAAAGAGCCAATAAGCCCA-3′) and G9+B (5′-TAGTCCTCAAGCAAGTCCTC-3′).

In order to identify the chlamydial species, the samples that had tested positive to the real-time PCR were characterized by sequencing a 16S rRNA gene portion of 278 bp with the primers set described by Vicari et al. [43 ]. The FastStart™ Taq DNA Polymerase kit (Roche Diagnostics, Mannheim, Germany) was used with 5 µL of extracted DNA as template in a final volume of 25 µL with the following final concentrations: 2.5 mM of MgCl₂, 0.2 mM of each dNTPs and 0.4 µM of each primer. After the initial denaturation step at 95 °C for 15 min, a touchdown protocol was applied to 20 cycles at 95 °C for 30 s, a decreasing annealing temperature from 65 °C to 55 °C for 30 s and 72 °C for 30 s, followed by 25 cycles with the same conditions and annealing step at 55 °C. A final extension step at 72 °C for 5 min was performed. PCR-amplified segments were purified with ExoSAP-IT Express PCR Product Cleanup (Applied Biosystems, Foster City, CA, USA) and sequenced with Brilliant Dye™ Terminator Cycle Sequencing kit Big Dye^® Terminator v3.1 Cycle Sequencing (Nimagen, Nijmegen, The Netherlands) on a the ABI PRISM^® 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
Sequencing data were subjected to BLAST search against the NCBI database to identify the most related sequences.

Tail snip samples were collected from pups before P3. DNA was extracted using the reagents and protocol of the QIAGEN QIAamp DNA Mini Kit 250 (catalog 51306). After extraction, PCR was performed using 0.25 μL mixture of primers 5′ AGA AAA GTT CTT CAC TGG TTG ACA 3′ (forward) and 5′ CAT CAT CAT CCC TGG TTC CTG 3′ (reverse), 6.25 μL Quanta Bio’s AccuStart GelTrack PCR SuperMix (catalog 95136), and 4 μL DDI H₂O. The reaction was run at 95°C for 2 minutes, followed by 35 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 minute, followed by 72°C for 1 minute. After completion of PCR, excess nucleotides were removed using 4 μL of applied biosystems ExoSAP-IT Express PCR Product Cleanup (catalog 750011). The reaction was run at 37°C for 1 minute, followed by 37°C for 14 minutes, and ending with 80°C for 15 minutes. Samples were sent to Eton Biosciences with the forward primer for sequencing to analyze the C1186T loci of exon 12 of the Tmem67 gene.

PCR products were purified with ExoSAP-IT Express PCR Product Cleanup (Applied Biosystems, Waltham, MA, USA). Sequence reaction was performed by 2 min/96 °C pre-incubation followed by 35 cycles with denaturation at 96 °C for 30 s, annealing at 50 °C for 15 s and extension at 60 °C for 3 min. Sequence products were purified with AutoSeq G-50 Dye Terminator Removal kit (Illustra, GE Healthcare, Chicago, IL, USA). The purified sequence products were dissolved in 10 μL Hi-Di Formamide (Applied Biosystems, Waltham, MA, USA) and run on the 3500 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA).

DNA extraction of six individuals of Mayaweckeliatroglomorpha sp. n., one M.cenoticola specimen and three T.cernua individuals (two from Yucatán state and one from Quintana Roo state, see Table 1 for sample data) was performed using QIAamp DNA Microkit (Qiagen), following the manufacturer’s instructions. Only a few pereopods were used for DNA isolation of each animal. For PCR amplification of mitochondrial cytochrome c oxidase subunit I (COI) we used the primer pair LCO 1490 and HCO 2198 (Folmer et al. 1994 (link)). PCR reactions (25 µl) were obtained by mixing 13.85 µl mQ water, 2.5 µl 10X PCR buffer, 2.5 µl dNTP mix (2mM), 1.5 µl of each primers (5µM), 0.15 µl Fermentas Dream Taq (5U/ µl) and 3 µl DNA extract. PCR temperature conditions were as follows: initial denaturation for 3 min at 94 °C, denaturation for 45 sec at 94 °C, hybridization for 45 sec at 48 °C, and polymerization for 1 min at 72 °C. After thirty cycles a final extension for 3 min at 72 °C was added. PCR products were cleaned using Exo SAP-IT Express PCR Product Cleanup (Affymetrix) according to manufacturer’s instructions. The fragments were sequenced in both directions using PCR amplification primers with an ABI 3130 sequencer. 638 bp COI barcode sequences have been uploaded to NCBI GenBank database. Accession numbers are MF589975–MF589984 (see Table 1).

Twenty-eight of the 66 isolates were sequenced and identified in the present study at the genus and species level via 16S rRNA gene sequence analysis. Genomic DNA was extracted from each strain as described by Seldin and Dubnau (1985) (link). The primers 8F (5′-AGA GTT GTA TCA TGG CTC AG-3′) and 1492R (5′-CGGTTA CCTTGT TAC GACTT-3′) were used to amplify the 16S rRNA gene. 2.5 mM MgCl₂, 0.5 mM of each dNTPs, 1 U of Taq DNA polymerase, 1 × GoTaq^® Flexi Buffer (Promega, Madison, United States) and 0.2 mM of each primer. PCR amplification was performed in a final volume of 25 μL containing 1 × Green GoTaq^® Flexi Buffer (Promega^® ; Madison, United States), 2.5 mM MgCl₂, 0.5 mM dNTPs, 0.2 μM each primer, 1 U of GoTaq^® (Promega), and 1 μL of template DNA (50–100 ng). PCR products were purified with ExoSAP-IT^® Express PCR Product Cleanup (Affymetrix, Santa Clara, CA) and sequenced using an Applied Biosystems 3500 Serial Genetic Analyzer (Applied Biosystems^®) at the Evandro Chagas Technological Innovation Center (Belém, Brazil). The sequences were compared to those in the GenBank database using the BLAST algorithm (National Center for Biotechnology Information, Maryland, United States).