A total of 12 DNA samples (for all three locations) and four cDNA samples (for the S2 location only) were amplified in 50-μL reaction systems using the primer sets of barcoded Pro341F (5′-XXXXXXXXCCTACGGGNBGCASCAG-3′, XXXXXXXX denotes the barcode sequence) and Pro805R (5′-GACTACNVGGGTATCTAATCC-3′) (Bourtzis et al., 2014 (link)) under the aforementioned conditions. For each sample, three replicates of both 16S rDNA and 16S cDNA amplicons were pooled and purified using an AxyPrep DNA Gel Extraction Kit (Axygen, Tewksbury, MA, United States). The amplicon DNA concentrations were measured using a Qubit dsDNA HS Assay Kit with a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, United States). Based on the quantification results, the cleaned amplicons were mixed at equimolar ratios for library construction. Using a KAPA LTP Library Kit (KAPA Biosystems, Boston, MA, United States), end-repairing, amplicon A-tailing, and adaptor ligating were carried out according to standard protocols. After ligation of the sequencing adaptors, each composite library was cleaned again with an AxyPrep DNA Gel Extraction Kit (Axygen, Tewksbury, MA, United States). Next-generation sequencing was performed on an Illumina MiSeq platform using the 2 × 300 protocol.
Axyprep dna gel extraction kit
Manufactured by Corning
Sourced in United States, China, Spain
The AxyPrep DNA Gel Extraction Kit is a laboratory tool designed to efficiently extract and purify DNA fragments from agarose gels. The kit provides a reliable and streamlined process for the recovery of DNA samples, making it a useful tool for various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Quantifluor st, by Promega (356 mentions)
Miseq platform, by Illumina (322 mentions)
E z n a soil dna kit, by Omega Bio-Tek (169 mentions)
Quantus fluorometer, by Promega (133 mentions)
Nanodrop 2000 uv vis spectrophotometer, by Thermo Fisher Scientific (120 mentions)
Miseq pe300 platform, by Illumina (94 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (84 mentions)
Abi steponeplus real time pcr system, by Thermo Fisher Scientific (48 mentions)
E z n a stool dna kit, by Omega Bio-Tek (36 mentions)
Qiaamp dna stool mini kit, by Qiagen (35 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (34 mentions)
Fastdna spin kit for soil, by MP Biomedicals (28 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (24 mentions)
Hipure stool dna kit, by Magen Biotechnology Co (23 mentions)
Transstart fastpfu dna polymerase, by Transgene (22 mentions)
Qubit 2, by Thermo Fisher Scientific (21 mentions)
Hipure soil dna kit, by Magen Biotechnology Co (20 mentions)
Nanodrop, by Thermo Fisher Scientific (20 mentions)
Quantifluor st fluorometer, by Promega (16 mentions)
Fastpfu polymerase, by Transgene (16 mentions)
1 108 protocols using axyprep dna gel extraction kit
1
Amplifying and Sequencing 16S rDNA and 16S cDNA
2
Microbial 16S rRNA Gene Amplification and Sequencing
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The microbial cells were scraped from the collected filters using sodium phosphate buffer. Total DNA was extracted using a FastDNA Spin Kit for Soil (MP Biomedicals, United States) and detected using 1% agarose gel electrophoresis. The V3–V4 region of the microbial 16S rRNA gene was amplified with primers 338F (5′-barcode-ACT CCT ACG GGA GGC AGC AG-3′) and 806R (5′-GGA CTA CHV GGG TWT CTA AT-3′). Amplicons were purified using an AxyPrepTM DNA Gel Extraction Kit (AXYGEN, United States) and quantified using a QuantiFluor-ST Fluorometer (Promega, United States), where the barcode is an eight-base sequence unique to each sample. PCR reactions were performed in triplicate in a 20-μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA.
3
Isolation and Sequencing of RSMV Genome
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Ten pairs of specific primers (Table 2 ) were designed according to the reported RSMV Guangdong isolate (GenBank Accession No. KX525586.2) using the PrimerSelect program in DNASTAR 7.1 (Lasergene, United States) so as to amplify the complete genome sequence that including the 3′-terminal and 5′-terminal sequences. Total RNAs from RSMV-infected samples were used as a template and a one-step RNA PCR kit (TaKaRa, Dalian, China) was used according to manufacturer’s instructions. The RT-PCR program was 50°C for 30 min, 94°C for 2 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and 72°C for 10 min. The PCR products were analyzed by electrophoresis in 1.2% agarose gel (stained with 5 μl/100 ml GoldView), purified using the AxyPrePTM DNA gel Extraction Kit (AxyGEN), and sequenced directly in both directions with three replicates (Shanghai Biotech, Shanghai, China). Sequence assembly and analysis was performed using the SeqMan program in DNASTAR 7.1 (Lasergene), and the whole genome sequence of each isolate was obtained (Supplementary Table S7 ) and submitted to the Genbank. The accession numbers of each sequenced RSMV isolate are listed in Table 3 .
4
Phylogenetic Analysis of Nucleotide Sequences
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The DNA fragments were purified with a AxyPrepTM DNA Gel Extraction Kit (Axygen, Union City, CA, USA), cloned into pGEM-T Easy vector (Promega, Madison, WI, USA) and transformed for sequencing using BigDye Terminator Mix (GenScript, Nanjing, China). The nucleotide sequences obtained in this study were compared with previously published sequences deposited in GenBank by a BLASTn search or by using the ClustalW multiple alignment algorithm in the MegAlign program of the Lasergene 7.1 software package (DNAStar, Madison, WI, USA). The phylogenetic trees were inferred by using the neighbor-joining (NJ) method with the Kimura two-parameter model, and the bootstrap test was replicated 1000 times [12 (link)].
5
Constructing AhHsp70p-Luciferase Reporter Plasmid
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To generate an AhHsp70p target luciferase reporter, we designed five deletion AhHsp70p specific primers (Table 1 ) using the CE V1.04 software. A KpnI restriction site sequence with approximately 15–25 bp homologous to the vector region was added to the 5ʹ end of the forward primer and an XhoI restriction site with approximately 15–25 bp homologous to the vector was added to the 5ʹ end of the reverse primer. The target fragment was obtained by PCR amplification using genomic DNA as a template. PCR amplification reactions were performed using a total reaction volume of 25 µL comprising 2.5 µL of 10× PCR buffer (10 µM), 0.5 µL of dNTPs (2.5 mM), 0.5 µL of Taq DNA polymerase (TransGen Biotech, China), 1 µL of each gene-specific primer pair, 0.5 µL of genomic DNA template, and 19 µL ddH2O. The PCR products were purified using an AxyPrepTM DNA Gel Extraction Kit (Axygen) and cloned into a pEASY-T3 vector (TransGen, Beijing, China). The veracity of the plasmids was confirmed using commercial sequencing (Sangon Biotech, Shanghai, China).
6
Identification of ISWI and BRM Genes in T. absoluta
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The homologous chromatin-remodelling ATPase genes ISWI and BRM from Bombyx mori (XP_012547334.1 and XP_037871728.1) and Drosophila melanogaster (NP_523719.1 and NP_536745.4) were used in blastp and tblastn to query T. absoluta transcriptome datasets. Specific PCR primers were designed on the basis of the foregoing fragments (Table 1 ). PCR amplifications were conducted using FastPfu DNA Polymerase (TransGen, Beijing, China). The amplified fragments were purified with an AxyPrepTM DNA Gel Extraction Kit (Axygen, West Orange, NJ, USA) according to the manufacturer’s instructions, cloned into a pEASY-Blunt Vector (TransGen, Beijing, China), and sequenced.
7
Cloning of TRPA1, Painless, and Pyrexia
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The full length cDNAs were obtained using a Taq DNA Polymerase amplification kit (TransGen, Beijing, China) according to the manufacturer’s instructions. The TRPA1, Painless, and Pyrexia homologous genes of B. mori (BAO53207.1, BAO53208.1, and NP_001296536.1) and D. melanogaster (NP_001261602.1, NP_001261176.1, and NP_612015.1) were used to query the transcriptome dataset of T. absoluta by blastp and tblastn. According to the corresponding sequences of T. absoluta, primers were designed by primer 5.0 (Table 1 ). The amplified fragments were purified using an AxyPrep TM DNA Gel Extraction Kit (Axygen, West Orange, NJ, USA). Finally, the distinct single-band amplification products were cloned into the pEASY-T3 vector (Transgen) and sequenced.
8
ZmGAPP Promoter Isolation and Characterization
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The 5' flanking sequence of ZmGAPP was retrieved from the NCBI High Throughput Genomic Sequences Database of Zea mays using its full-length cDNA sequence (GenBank accession no. EF051578) as query. The forword and reverse primers (named pZmGAPPFR, Table 1 ) were designed according to the ZmGAPP sequence and its 5' flanking sequence. The 1584-bp fragment (–1468 to +116 bp; the “A” of the translation start codon “ATG” of ZmGAPP was designated as “+1”) was amplified from maize genomic DNA with the pZmGAPPFR primers (Table 1 ). The PCR conditions were as follows: initial denaturation at 95°C for 5 min followed by 35 cycles of 95°C 1 min, 55°C 1 min, and 72°C 2 min, and then final extension at 72°C for 7 min. The PCR products were excised from a 1% agarose gel and purified by AxyPrepTM DNA Gel Extraction Kit (Axygen Scientific, Inc, China). Then the fragment (–1468 to +116 bp) were cloned in the pGEM-T® Easy cloning vector (Promega, USA) following the manufacturer's instructions and confirmed by sequencing. Finally, a 1468-bp fragment upstream of the translation start codon of ZmGAPP was isolated by PCR amplification using D1 primers (Table 1 ) and considered as the full-length promoter.
9
Cloning and Sequencing of Bemisia tabaci Dnmt1 Gene
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Primers were designed based on the transcriptome information of B. tabaci, and were used to amplify partial segments of the BtDnmt1 gene. Next, rapid amplification of cDNA ends (5′- and 3′-RACE) was performed to obtain the full-length cDNAs using a SMART RACE cDNA amplification kit (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions. The gene-specific primer sets (Table 1 ) were designed based on the B. tabaci transcriptome information from the NCBI website (http://www.ncbi.nlm.nih.Gov ). The amplified fragments were purified using an AxyPrepTM DNA Gel Extraction Kit (Axygen, West Orange, NJ, USA). Finally, the distinct single-band amplification products were cloned into the pEASY-T3 vector (Transgen, Beijing, China) and sequenced.
10
COI gene sequencing protocol
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Approximately half of the COI coding region (758 bp) was polymerase chain reaction (PCR) amplified in all samples using universal primers C1-J-2195 (5′-TTGATTTTTTGGTCATCCAGAAGT-3′) and TL2-N-3014 (5′-TCCAATGCACTAATCTGCCATATTA-3′) (
Simon et al. 1994
). Reaction conditions consisted in an initial denaturation of 94°C for 5', followed by 35 cycles of 94°C for 45", 50°C for 1' and 72°C for 1'30", and a final extension at 72°C for 10'. PCR products were gel purified using the AxyprepTM DNA Gel Extraction Kit (AXYGEN, Union City, CA) and directly sequenced in an ABI 3730 XL DNA analyzer. In total, 77 new sequences were deposited in GenBank under accession numbers KC123182–KC123194, KC113525–KC113578, HQ916813–HQ916821, and KF534718 (
Table 1 ).
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Approximately half of the COI coding region (758 bp) was polymerase chain reaction (PCR) amplified in all samples using universal primers C1-J-2195 (5′-TTGATTTTTTGGTCATCCAGAAGT-3′) and TL2-N-3014 (5′-TCCAATGCACTAATCTGCCATATTA-3′) (
Simon et al. 1994
). Reaction conditions consisted in an initial denaturation of 94°C for 5', followed by 35 cycles of 94°C for 45", 50°C for 1' and 72°C for 1'30", and a final extension at 72°C for 10'. PCR products were gel purified using the AxyprepTM DNA Gel Extraction Kit (AXYGEN, Union City, CA) and directly sequenced in an ABI 3730 XL DNA analyzer. In total, 77 new sequences were deposited in GenBank under accession numbers KC123182–KC123194, KC113525–KC113578, HQ916813–HQ916821, and KF534718 (
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