Pcr purification kit

For DNA extraction wild type W0 and its mutant M0, mycelium was cultured on potato dextrose broth (PDB) and incubated for 48 h at 28 °C under shaking condition (200 rpm). Mycelia was harvested and washed with sterilized water and ethylenediamine tetra acetic acid (EDTA). The DNA was extracted by cetyl trimethylammonium bromide (CTAB) method [6 ]. The specific primers β-TUB (F-5′-TGAAGGTATGGACGAGAT-3′) (R-5′-GCATCCTGGTATTGTTGA-3′) under accession number (XM_001560987.1) and α-TUB (F-5′GTTGGAGTTCTGTGTCTA-3′) (R-5′GTGGTCAAGATGGAGTTA-3′) under accession number (XM_001555875.1) were used to amplify the complete coding sequence (CDS) of two Tubulin genes bctubA and bctubB. Three biological replicates of each strain used for DNA extraction and the PCR reactions were conducted three times independently for each sample. The amplified PCR products were purified using a PCR Purification Kit (TIANGEN, Beijing, China), ligated into the pMD18-T Vector (TaKaRa Biotechnol. Co., Ltd., Dalian, China), and then sequenced by Sangon (Guangzhou, China). The exon sequences of the bctubA and bctubB genes were translated into amino acid sequences and aligned using DNAMAN8.0 software (Lynnon Biosoft, Quebec, Canada) to check the mutation point.

The occurrence of β-lactamase genes (bla_TEM, bla_SHV, bla_CTX−Ms), plasmid-mediated AmpC β-lactamase (bla_CMY−2) and carbapenemase genes (bla_KPC−2, bla_NDM−1, and bla_OXA−48) among ESBL producers were determined by PCR and sequencing using specific primers (Table S2). The PCR products were purified using a PCR Purification Kit (TianGen, Beijing, China), and then the amplified products were sequenced by Sangon Biotech (Shanghai, China). DNA Sequences were compared with known sequences available from the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1997 (link)). Additionally, all ESBL producers were screened for the presence of PMQR genes (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6')-Ib-cr, oqxAB, and qepA) as described previously (Liu et al., 2012 (link); Xu et al., 2015 (link)). E. coli J53 strains containing pMG252, pMG298, pMG306, and pMG298 were used as positive controls for qnrA, qnrB, qnrS, and aac(6′)-Ib-cr genes, respectively. E. coli J7261205 (pSTVqepA) and S5314175 were included as positive controls for qepA and oqxAB, respectively. The positive control strain for qnrC was not available.

Total RNA samples of M. abei were submitted for diagnostic analysis of CMNV by reverse transcription nested PCR (RT-nPCR). Firstly, cDNA were synthesized from total RNA by using the SMART^® MMLV Reverse Transcriptase (TAKARA) with the primer of CMNV-7R1 according to the recommended procedures. The first step PCR were conducted by using CloneAmp HiFi PCR Premix (Takara) with the primers sets of CMNV-7F1/R1 and annealing at 50°C. The second step PCR was carried out according to our previous report with a minor modification (Zhang et al., 2014 (link)). The expected CMNV target fragments would be 619 bp amplicon and 413 bp amplicon after the first and second step of the PCR amplifications, respectively. The PCR products were purified by a PCR purification kit (Tiangen, Beijing, China) and then subjected for sequencing by the commercial sequencing company of Shanghai SANGAN Chemical Trading Co. Ltd.

To create FLAG-AQP0 and Myc-AQP0 fusion proteins, the PCR products of WT-AQP0 and Y219*-AQP0 were digested with BamHI and XhoI restriction enzymes, respectively, purified with a PCR purification kit (Tiangen Biotech, Beijing, China), and subsequently cloned into the digested mammalian expression vector, pCMV-3Tag-6(Agilent Technologies, Shanghai, China), which contained an N-terminal triple FLAG epitope tag;and pCMV-3Tag-7 (Agilent Technologies, Shanghai, China), which contained an N-terminal tripleMyc epitope tag. All of the constructs were verified by direct sequencing.

Three-day old mycelium from resistant mutants and wild type strains were cultured in PDB medium (Hopebio; Qingdao, China) and collected for extraction of DNA using the CTAB method [53 (link)]. The specific primers FVER_05465F (5’-AGCGGCCAGTTAT TTCAGCA-3′), FVER_05465R (5’-GCCGATTTCTCTCCTCCTTCTC-3′), FVER_0 9254F (5’-TCCAATCCCTCTAGCCCTCG-3′) and FVER_09254R (5’-TCCTCGACA ACTTCACCACG-3′) were designed to amplify complete coding sequence (CDS) of Tub2 gene based on the genome sequence of F. verticillioides CNO-1. FPRO_14041 and FPRO_07779 are derived from the genome of F. proliferatum YN41 and the amplified primers are identical to the FVER_0 9254 and FVER_05465, respectively. Three biological replicates of each strain used for DNA extraction and the PCR reactions were conducted three times independently for each sample. The amplified PCR products were purified using a PCR Purification Kit (TIANGEN; Beijing, China), ligated into the pMD18-T Vector (TaKaRa Biotech; Dalian, China), and then sequenced by Sangon (Guangzhou, China). The exon sequences of the Tub2 gene were translated into amino acid sequences and aligned using DNAMAN8.0 software (Lynnon Biosoft; USA).

The double blood bags used blood collection were obtained from Suzhou Laishi Transfusion Equipment Co., Ltd (Suzhou, China). The complete Freund’s adjuvant, incomplete Freund’s adjuvant and Amicon UltraCentrifugal Filter Units were procured from Sigma Aldrich (St. Louis, MO, USA). All the restriction enzymes utilized in the study were procured from New England Biolabs (Beijing) LTD (Beijing, China). The 96-well microplates were purchased from Corning (New York, NY, USA). The PCR Purification Kit, Gel Extraction Kit and TIANprep Mini Plasmid Kit used for plasmid preparation were obtained from TIANGEN (Beijing, China). All the reagents utilized in this study were analytical grade, unless otherwise indicated.

SL-44 was characterized in morphological, physiological, and biochemical characterization and then compared with standard species using Bergey's Manual of Determinative Bacteriology. The bacterial isolate was genetically identified by 16S rDNA gene sequence analysis; the PCR amplification was performed using universal primers according to the method of Yao et al. [18 (link)]. The amplification products were purified using a PCR purification kit (Tiangen, China) and sequenced.