Total gut, liver, and kidney DNA was extracted using a QIAamp-DNA Stool Kit (Qiagen, Hilden, Germany). Primers for amplification of genes are listed in Table S1. Amplified sequences were cloned into pMD19-T plasmids (Takara, Dalian, China), to perform a 10-fold dilution and generate a standard curve for calculation of the bacterial load [128 (link), 129 (link)]. The values obtained from bacterial copies were calculated relative to the weight of the tissues.
Pmd19 t plasmid
Manufactured by Takara Bio
Sourced in China, Japan
The PMD19-T plasmid is a cloning vector used for the insertion and propagation of DNA fragments in bacterial hosts. It contains a multiple cloning site, an antibiotic resistance marker, and an origin of replication for plasmid maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Ampicillin, by Avantor (4 mentions)
Kanamycin, by Avantor (4 mentions)
Ex taq dna polymerase, by Takara Bio (4 mentions)
Chloromycetin, by Avantor (4 mentions)
Plasmid mini prepare kit, by Corning (4 mentions)
Dna mini kit, by Corning (4 mentions)
T4 dna ligase, by Takara Bio (4 mentions)
Escherichia coli dh5α, by Takara Bio (4 mentions)
Isopropyl β d thiogalactopyranoside, by Avantor (4 mentions)
Qiaamp dna stool kit, by Qiagen (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
La taq, by Takara Bio (1 mentions)
Puc57 plasmid, by Tsingke (1 mentions)
Cfx connect, by Bio-Rad (1 mentions)
Tianamp bacteria dna kit, by Tiangen Biotech (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Image analyzer, by Bio-Rad (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Escherichia coli bl21 de3, by Transgene (1 mentions)
His tag protein purification kit, by Beyotime (1 mentions)
19 protocols using pmd19 t plasmid
1
Quantification of Gut Microbiome DNA
2
Identifying BLG-Targeted Cell Clones
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Drug-resistant cell clones derived from the transfected cell populations were collected by trypsinization, and 70% of which were plated in serum-containing culture medium and expanded. The remaining clones were resuspended in 20 μL of PCR-compatible lysis buffer (10 mM of Tris-HCl, pH 8.5; 50 mM of KCl; 1.5 mM of MgCl2; 0.5% NP-40; 0.5% Tween-20; 400 g/mL of proteinase K) for PCR analysis. The lysates were incubated at 65 °C for 60 min and then at 95 °C for 15 min.
To distinguish the BLG-targeted cell clones, 2 μL of the DNA lysate was added to a PCR reaction with PCR primers for 5′ (or 3′) junction PCR and subjected to PCR with La Taq (TaKaRa) for 30 cycles (95 °C, 30 s; 60 °C, 30 s; 72 °C, 90 s). Subsequently, 3′ (or 5′) junction PCR was performed on the 5′ (or 3′) positive clones to confirm the correct targeting events. Clones that passed both tests were expanded, and their genomic DNA was extracted with TIANamp Genomic DNA Kit (Tiangen Biotech) to confirm the targeting events by PCR analysis as described above. The PCR products were cloned into pMD19-T plasmids (TaKaRa) for sequencing (GenScript Co., Ltd.).
To distinguish the BLG-targeted cell clones, 2 μL of the DNA lysate was added to a PCR reaction with PCR primers for 5′ (or 3′) junction PCR and subjected to PCR with La Taq (TaKaRa) for 30 cycles (95 °C, 30 s; 60 °C, 30 s; 72 °C, 90 s). Subsequently, 3′ (or 5′) junction PCR was performed on the 5′ (or 3′) positive clones to confirm the correct targeting events. Clones that passed both tests were expanded, and their genomic DNA was extracted with TIANamp Genomic DNA Kit (Tiangen Biotech) to confirm the targeting events by PCR analysis as described above. The PCR products were cloned into pMD19-T plasmids (TaKaRa) for sequencing (GenScript Co., Ltd.).
3
Characterization of Lamprey TLR14 Gene
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The preliminary sequence of LmTLR14d was obtained by the BLAST method from the genomes of sea lamprey (https://genomes.stowers.org/organism/Petromyzon/marinus ) and Japanese lamprey (http://jlampreygenome.imcb.a-star.edu.sg/blast/ ). The primers were designed by using highly conserved homologous genes from vertebrates (Supplementary Table 1
4
Transgenic Silkworm Protocol: Nistari Strain
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The multivoltine and monophagous B. mori strain, Nistari, was used as the basic transgenic strain. Silkworm larvae were fed fresh mulberry leaves and maintained at 25 °C under standard conditions [43 (link)]. The pMD19T plasmid was purchased from TaKaRa (Dalian, China). The PUC57 plasmid was purchased from TSINGKE Biological Technology (Nanjing, China). The pET32a plasmid, pET24b-BmSuc1 recombinant plasmid, and anti-BmSUC1 polyclonal antibody were maintained in our laboratory. The plasmid PXL-IE1-DsRed2-U6-U6 (Sal I, Nhe I), containing the B. mori U6 promoter, was used to construct the sgRNA expression vector.
5
Quantify Geobacter species in co-cultures
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The primers used for bacterial quantification are given in Supplementary Table 1 . After fumarate was consumed completely, the co-cultures were shaken vigorously and a 2-ml culture medium was used to extract the total genomic DNA with the TIANamp Bacteria DNA Kit (Tiangen, Beijing, China). The unique DNA fragments of G. metallireducens and G. sulfurreducens were amplified by primer pairs qGmetf/qGmetr and qGsulff/qGsulfr, respectively. The two DNA fragments were connected with pMD19-T plasmid (Takara, Kusatsu, Japan) for T-A cloning and then the plasmids were verified by primer pair M13f/M13r. The plasmids were extracted with the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific, Waltham, MA, United States) and then gradient diluted, which was used for the standard solution of quantitative real-time PCR (qPCR). Unique DNA fragments and standard solution were simultaneously quantified with a real-time quantitative PCR detection system (CFX Connect, Bio-Rad, Hercules, CA, United States) to obtain the number of G. metallireducens and G. sulfurreducens in the co-cultures (Huang et al., 2020 (link)).
6
PCR Product Sequencing Protocol
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The PCR products were examined in agarose gel using an Image Analyzer (Bio-Rad, USA) after electrophoresis. PCR products less than 800 bp were sequenced directly, and the PCR products more than 800 bp were firstly purified using a Gel Extraction Kit (Qiagen, USA) according to the manufacturers’ protocols, then cloned into pMD19-T plasmid (TaKaRa, China) and transformed into E. coli JM109 competent cells. For each amplicon, the positive inserts were confirmed by PCR, and three positive clones were sequenced with the universal M13 forward and reverse primers in cloning vector. All the sequencing procedures were performed by Sangon Biotechnology Company in China.
7
Recombinant On-LECT2 Protein Production
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The specific primers, namely, On-LECT2 recombinant protein-S (rOn-LECT2-S) and rOn-LECT2-A, with the restriction sites Bam HI and Xho I were designed (Table S1
8
Quantitative real-time PCR protocol
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Total genomic DNA was extracted as described above. qPCR was performed using a Bio-Rad real-time PCR detection system (CFX Maestro 1.1 or 3.0; Bio-Rad Inc., Hercules, CA, USA) and SsoFast EvaGreen supermix (Bio-Rad Inc.) according to the manufacturer’s instructions. Primer sequences for target genes are shown in Table S3 in the supplemental material. The reaction protocol for all genes was as follows: 5 min at 95°C followed by 40 cycles of 10 s at 95°C and 30 s at 60°C. A dilution gradient of a pMD19-T plasmid (TaKaRa, Dalian, China) containing the target genes was used to obtain a standard curve, according to which the absolute expression levels of target genes were calculated.
9
Cloning and Verification of Murine Akirin1
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According to the NCBI reference sequence of murine Akirin1 (GenBank accession no. NM_023423.3), the cDNAs encoding Akirin1 were amplified by PCR with P1 (Table 1 ). These cDNAs were cloned into the pMD19-T plasmid (TaKaRa, Japan) and named pMD19-T-Akirin1. The entire coding region of murine Akirin1 was digested from pMD19-T-Akirin1 plasmid and then inserted into the PEGFP-N1 plasmid via EcoRI and BamHI sites. After sequencing (Applied Invitrogen, China), the second recombinant plasmid was confirmed and named as pEGFP-N1-Akirin1. To evaluate its quality, the second recombinant plasmid was extracted and double-digested by EcoRI and BamHI (Beyotime Biotech, China).
10
Molecular Characterization of CmBBX8 Transcription Factor
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RNA was extracted from cv. ‘Yuuka’ leaf tissue using the TRIzol reagent (Invitrogen, New York). The CmBBX8 ORF sequence was amplified using the primer pair CmBBX8‐F/‐R (annexed table), and the resulting amplicon was inserted into the pMD19‐T plasmid (Takara, Beijing, China) for the purpose of sequencing. The domain content of the CmBBX8 sequence was inferred by querying the Conserved Domains database (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ). The sequences of BBX8 homologs were recovered from GenBank and used to construct a multiple sequence alignment with the help of Clustal X v2.1 software (Aiyar, 2000 ). The phylogeny of a set of BBX sequences was derived using MEGA v5.05 software (Tamura et al., 2007 ), applying the neighbour‐joining algorithm with 1000 bp replications.
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