Gf 1 ambiclean kit

The specific amplicon was purified with the GF-1 AmbiClean Kit (Vivantis, Malaysia). The products were subsequently cloned into the pGEM-T Easy vector (Promega, USA) according to the manufacturer′s instructions. The recombinant plasmids were transformed into E. coli (DH5α) competent cells and screened for positive colonies. Plasmids were extracted using the GF-1 Plasmid DNA Extraction Kit (Vivantis, Malaysia). All sequencing reactions were performed on both strands using the sequencing service from Macrogen Inc., Seoul, Korea.

A yeast strain that produced the largest amount of ethanol from xylose was identified by nucleotide sequencing of the D1/D2 domain from the large-subunit (LSU) rRNA gene. Genomic DNA was extracted as described previously [41 (link)]. Forward primer NL-1 (5′-GCATATCAATAA GCGGAGGAAAAG-3′) and reverse primer NL-4 (5′-GGTCCGTGTTTCAAGACGG-3′) were used to generate the PCR product of the D1/D2 domain [42 ]. The PCR product was checked by agarose gel electrophoresis and purified by using a GF-1 AmbiClean Kit (Vivantis, Malaysia). The sequences of the PCR product were determined by 1st BASE (Selangor, Malaysia) and examined via a BLAST homology search [43 (link)]. The evolutionary history was inferred using the Neighbor-Joining method [44 (link)]. The evolutionary distances were computed using the Kimura 2-parameter method [45 (link)]. Evolutionary analyses were conducted using MEGA7 software [46 (link)].

DNA extraction was performed following the method of Kudo et al. (1998) (link). Genomic DNA was used as the template for polymerase chain reaction (PCR) following the method of Lee et al. (2014) (link). 27F (5′ AGAGTTTGATCMTGGCTCAG 3′) and 1492R (5′ TACGGYTACCTTGTTACGACTT 3′) primers were used to amplify 16S rRNA gene. The PCR amplicons were purified by GF-1 AmbiClean Kit (Vivantis^®) following the manufacturer instruction. The 16S rRNA sequencing was performed by commercial service at First BASE Laboratories Sdn Bhd, Malaysia. Identification of all actinobacterial isolates was achieved by BLAST analysis of 16S rRNA gene sequences using EzBiocloud database¹. Neighbor-joining phylogenetic tree was constructed using BioEdit Sequence Alignment Editor version 7.2 and MEGA7 (Kumar et al., 2016 (link)). Tree topology was evaluated using the bootstrap resampling method at 1000 bootstrap. Strain CMU55-4 was also identified based on whole genome sequence analysis. The ANI value was calculated and compared in JSpeciesWS (Ritcher et al., 2016 (link)), web server tool, using ANI-Blast (ANIb) and ANI-MUMmer (ANIm) algorithms (Ritcher and Rosselló-Móra, 2009 (link)) within the web service. The Genome-to-Genome Distance Calculator (GGDC 2.1) with the BLAST + method (Meier-Kolthoff et al., 2013 (link)) was used to evaluate the digital DNA-DNA hybridisation (dDDH).

After obtaining the adapted strains from the ALE process, nucleotide sequencing of the D1/D2 domain from the large subunit (LSU) rRNA gene was conducted to verify for confirming that such observations were not a result of unintentional contaminations. Genomic DNA extraction was performed as described previously [38 (link)]. The forward primer NL-1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and reverse primer NL-4 (5′-GGTCCGTGTTTCAAGACGG-3′) were utilized to generate the PCR product of the D1/D2 domain [39 ]. The PCR product was verified by agarose gel electrophoresis and purified by using a GF-1 AmbiClean Kit (Vivantis, Malaysia). The sequences of the PCR product were determined by 1st BASE (Selangor, Malaysia) and subjected to a BLAST homology search [40 (link)]. The determined nucleotide sequence was deposited in the DNA Data Bank of Japan (DDBJ).

Primers for amplification of the ompA gene were synthesised by Macrogen Co., Ltd., Korea. The gene was amplified using Taq DNA polymerase (PrimeSTAR GXL DNA Polymerase, Takara Bio. Inc., Japan) with the forward (OMPA-F, 5′-AGGATCCATGAAAAAAACAGCAATTGCATTGA-3′) and reverse (OMPA-R, 5′-TCTCGAGTTATTTGTTACCTTTAACAGCGATTTC-3′) primers; the sequences of these primers were modified from Gao et al. [49 ]. The PCRs contained 5 μl of 50 ng/μl genomic DNA, 10 μl of 5X PCR buffer, 5 μl of 2 mM dNTPs, 1 μl (5 U) of Taq DNA polymerase, and 5 μl of each of 2 μM forward and reverse primers in a total volume of 50 μl. The reaction mixtures were incubated for 30 cycles consisting of initial denaturation at 98 °C for 3 min; denaturation at 98 °C for 10 s, annealing at 55 °C for 15 s, extension at 68 °C for 1 min; and final extension at 68 °C for 3 min. The PCR products were purified using a GF-1 Ambiclean kit (Vivantis Technologies Sdn. Bhd., Malaysia), and the cleaned products were subjected to sequencing with both primers using an Applied Biosystems automatic sequencer (ABI 3730XL) (Macrogen Co., Ltd., Korea). The sequence chromatograms were checked for quality, and the ompA sequences were confirmed using the BLASTN program.

All positive amplicons were purified using the GF-1 Ambi Clean kit (Vivantis) according to the manufacturer’s instructions and sequenced in both directions on an ABI 3730xl DNA analyzer (Applied Biosystems). All obtained DNA sequences were assembled and edited using BioEdit (Alzohairy 2011 ). Edited sequences were assembled into a contig using SeqMan software (DNASTAR, Lasergene), and thus were subjected to BLASTn analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to find sequence similarity to known sequences. Phylogenetic analyses were performed using the maximum parsimony method (PAUP v. 4.0b1) and bootstrap analysis was calculated with 1,000 replicates.

Four selected PCR amplicons, two from the first sampling date (one lamb and one ewe) and two from the ninth sampling date (one lamb and one ewe), were purified with the GF-1 Ambi Clean kit (Vivantis, California, USA) according to the manufacturer’s instructions. Purified amplicons were sequenced in both directions using the same primers as for the A. ovis msp4-specific PCR amplifications (Table 1). The reaction was realized with a conventional Big Dye Terminator cycle sequencing ready reaction kit (Perkin Elmer, Applied Biosystems, Foster City, USA) and ABI3730XL automated DNA sequencer by Macrogen Europe (Amsterdam, The Netherlands). The evaluation of chromatograms was performed by using Chromas Lite v 2.01.
Multiple sequence alignments of the amplicons and nucleotides’ translation were performed using DNAMAN program (Version 5.2.2; Lynnon Biosoft, Quebec, Canada). A BLAST analysis was performed in GenBank to compare the published sequences with ours (http://blast.ncbi.nlm.nih.gov/, accessed on 24 June 2022) [25 (link)]. The DNAMAN program was used to build a phylogenetic tree based on the distance method using the neighbor-joining (NJ) algorithm of Saitou and Nei (1987) [26 (link)] with a bootstrap of 1000 iterations. Four msp4 amplicons were submitted to GenBank under accession numbers from MZ073666 to MZ073669.