Genephlow gel pcr kit

Genomic DNA was isolated using a PureLink Genomic DNA Mini Kit (Invitrogen, Waltham, MA, United States). The target regions for DNA sequencing were amplified by polymerase chain reaction (PCR) using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, United States) with specific primers, and the resulting PCR products were purified by a GenepHlow Gel/PCR kit (Geneaid, New Taipei City, Taiwan). A total of 0.2 μg of PCR product was then used for DNA sequencing using ABI PRISM BigDye Terminator Cycle Sequencing Kit v3.1 (first BASE, Singapore). The presence of insertion/deletion (INDEL) mutations in each target gene was determined by Inference of CRISPR Edits (ICE), the web-based analysis tool (available at https://ic-e.synthego.com/). Knockout (KO) score, which represents the proportion of cells that have either a frameshift or 21+ bp INDEL that are likely to generate a complete loss-of-function mutation, was determined.

Bacterial genomic DNA was extracted from liquid cultures with a Geneaid Genomic DNA Mini Kit, followed by PCR amplification with the universal eubacteria primers 27F and 1492R [16 (link)] in a final volume of 20 μL. Each 20-μL PCR mixture consisted of genomic DNA (50 ng), 10 μL of 2× Ready to Load PCR Master Mix (Cyrus Bioscience, MDBio. Inc., Annapolis, MD, USA), 0.5 μL of forward primer (10 µM), and 0.5 μL of reverse primer (10 µM). PCR was performed in a TProfesional Thermocycler^® (Analytik Jena AG, Jena, Germany) with the following cycling conditions: an initial denaturation at 95 °C for 2 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min. A final extension was performed at 72 °C for 10 min. PCR products were analyzed using 1% agarose gel electrophoresis. The size of the primer-amplified segment was expected to be 1465 bp. DNA fragments were purified using the GenepHlow™ Gel/PCR Kit (Geneaid) according to the manufacturer’s instructions. Sequencing of the PCR product was performed by Protech Technology Enterprise Co., Ltd. (Taipei, Taiwan). All the sequencing results were compared online with standard 16S rRNA sequences of bacteria in GenBank using nucleotide BLAST to identify close relatives.

The HAstV strains detected in this study were further characterized for their genotypes by nucleotide sequencing. The PCR products were purified by using GenepHlow™ Gel/PCR kit (Geneaid Biotech, Taiwan). Then, the purified PCR products were sequenced (First Base Laboratory SDNBHN Selangor Darul Ehsan, Malaysia). The obtained nucleotide sequences were analysed by comparing with those of the reference strains available in the GenBank database using the Basic Local Alignment Search Tool (BLAST) server (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The phylogenetic tree of the partial RdRp gene was constructed by using the MEGA X software based on the maximum likelihood method and selected the best-fit evolutionary model for the data set via Tamura-3-parameter model with 1000 replicates^{43 (link),44 (link)}.

Genomic DNA was isolated using a PureLink Genomic DNA Mini Kit (Invitrogen, Waltham, MA). The target regions for DNA sequencing were amplified by polymerase chain reaction (PCR) using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA) with specific primers, and the resulting PCR products were purified by a GenepHlow Gel/PCR kit (Geneaid, New Taipei City, Taiwan). A total of 0.2 μg PCR product was then used for DNA sequencing using ABI PRISM BigDye Terminator Cycle Sequencing Kit v3.1 (1st BASE, Singapore). The presence of insertion/deletion (INDEL) mutations in each target gene was determined by Inference of CRISPR Edits (ICE), the web-based analysis tool (available at https://ice.synthego.com/).

In order to screen for a potential recombinant strain, the ORF1a genotype of these 92 HAstV strains was determined by ORF1a nucleotide sequence analysis. The viral RNA genomes of these 92 HAstV strains were extracted using a viral nucleic acid extraction kit II (Geneaid Biotech, Taiwan) according to the manufacturer’s protocol. The cDNA was synthesized from the viral RNA genome using a RevertAid first-strand cDNA synthesis kit (Thermo Fisher Scientific, USA). The ORF1a region was amplified by using the primer sets listed in Table 2. The amplicons were detected by 1.5% agarose gel electrophoresis and purified using a GenepHlow gel/PCR kit (Geneaid Biotech). The purified PCR products were sequenced by using the Applied Biosystems Sanger sequencing kit (Thermo Fisher Scientific) with an automatic genetic analyzer (Applied Biosystems) provided by Apical Scientific Sdn. Bhd. (Malaysia) (formerly known as First BASE Laboratories Sdn. Bhd.). The sequences obtained were analyzed to assign the genotype of the ORF1a region by comparison with those of the reference strains available in the GenBank database using the Basic Local Alignment Search Tool (BLAST) server (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and phylogenetic analysis.

The viral RNA genomes of 92 archival HAstV strains were extracted using a Geneaid Viral Nucleic Acid Extraction Kit II (Geneaid, Taipei, Taiwan) according to the manufacturer’s protocol. The cDNA was synthesized from the viral RNA genome using RevertAid Fist Stand cDNA Synthesis Kit (Thermo Fisher Scientific, United States). Amplification of the ORF1b and ORF2 junction encompassing the 3′ end of ORF1b and 5′ end of ORF2 was performed by PCR and semi-nested PCR using the primer sets listed in Table 1. For classic HAstV, a semi-nested PCR was performed using two different alternative forward primers, SF0073 and SF0076-F, in combination with a set of outer reverse primer AHAstVR1 and inner reverse primer AHAstVR2, which generated PCR product sizes of 1104 and 708 bp, respectively. For novel HAstV-MLB, a conventional PCR was performed using forward primer SF0073 and reverse primer AHMLBR1 to generate a PCR product size of 926 bp. For novel HAstV-VA, a semi-nested PCR was performed using forward primer SF0076-F in combination with a set of outer reverse primer AHVAR1 and inner reverse primer AHVAR2, which generated a PCR product size of 987 bp. The PCR products were purified by using the GenepHlow™ Gel/PCR kit (Geneaid Biotech, Taiwan), and then, the purified PCR products were sequenced by First Base Laboratory SDNBHN Selangor Darul Ehsan, Malaysia.