The standard tet(X4) gene and other variants of tet(X) with their own promoters were amplified by PCR using KOD One PCR master blue mix (Toyobo, Osaka, Japan), except for tet(X6), tet(X6.2), and tet(X6.3), which have no function in pUC19. The primers and templates used for amplificon are shown in Table S2 in the supplemental material. Purified nucleic acid was cloned into plasmid pUC19. The constructed plasmids were then transformed into DH5α competent cells. The complete coding DNA sequences (CDSs) of tet(X6), tet(X6.2), and tet(X6.3) were cloned into pET23a (+) under the T7 promoter using NdeI and BamHI. Luria broth (LB) agar plates with ampicillin (100 μg/ml) and tigecycline (2 μg/ml) were used for transformant screening. Positive clones were confirmed by Sanger sequencing. E. coli DH5α carrying pUC19-tet(X4) was used as the engineered tet(X4)-mediated tigecycline-resistant strain. E. coli DH5α transformed with the blank plasmid pUC19 was considered the control for the engineered tigecycline-susceptible strain.
Kod one pcr master mix blue
Manufactured by Toyobo
Sourced in Japan
KOD One PCR Master Mix -Blue- is a ready-to-use solution for PCR amplification. It contains the high-fidelity KOD DNA polymerase, dNTPs, and reaction buffer.
Automatically generated - may contain errors
Lab products found in correlation
Revertra ace qpcr rt master mix, by Toyobo (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Clonexpress one step cloning kit, by Vazyme (1 mentions)
Fastgene rna premium kit, by Nippon Genetics (1 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Hiscript 2 q rt supermix for qpcr gdna wiper, by Vazyme (1 mentions)
Rapid taq master mix, by Vazyme (1 mentions)
Gotaq green master mix, by Promega (1 mentions)
Xbai, by Takara Bio (1 mentions)
Pb514b 2, by System Biosciences (1 mentions)
Superscript 3 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Xbai, by New England Biolabs (1 mentions)
High pure pcr template preparation kit, by Roche (1 mentions)
Zero blunt topo pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Isogen, by Nippon Gene (1 mentions)
Sybr green realtime pcr master mix, by Toyobo (1 mentions)
17 protocols using kod one pcr master mix blue
1
Cloning and Expression of Tetracycline Resistance Genes
2
Cloning and Expression of Tetracycline Resistance Genes
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The standard tet(X4) gene and other variants of tet(X) with their own promoters were amplified by PCR using KOD One PCR master blue mix (Toyobo, Osaka, Japan), except for tet(X6), tet(X6.2), and tet(X6.3), which have no function in pUC19. The primers and templates used for amplificon are shown in Table S2 in the supplemental material. Purified nucleic acid was cloned into plasmid pUC19. The constructed plasmids were then transformed into DH5α competent cells. The complete coding DNA sequences (CDSs) of tet(X6), tet(X6.2), and tet(X6.3) were cloned into pET23a (+) under the T7 promoter using NdeI and BamHI. Luria broth (LB) agar plates with ampicillin (100 μg/ml) and tigecycline (2 μg/ml) were used for transformant screening. Positive clones were confirmed by Sanger sequencing. E. coli DH5α carrying pUC19-tet(X4) was used as the engineered tet(X4)-mediated tigecycline-resistant strain. E. coli DH5α transformed with the blank plasmid pUC19 was considered the control for the engineered tigecycline-susceptible strain.
3
Optimized KOD One PCR Cloning
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All PCR reactions were performed using KOD One PCR Master Mix -Blue- (TOYOBO). Recombinational cloning was performed with the ClonExpress One Step Cloning Kit (Vazyme).
4
Gene Expression Analysis in Rat Spleens
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Total RNA was extracted from the spleens of 9-week-old WT F344/Jcl female, 16-week-old sKO female, and 14-week-old dKO female rats using a FastGene™ RNA Premium Kit (Nippon Genetics, Tokyo, Japan). First-strand cDNA was prepared from 1 μg of total RNA using ReverTra Ace® qPCR RT Master Mix (Toyobo, Osaka, Japan). The primers for Il2rg were 5’-CCGACCAACCTCACTATGCA-3’ and 5’-GATTCTCTGGAGCCCATGGG-3’ ; for Rag2, the primers were 5’-AAGGCAGCACAGACTCTGAC-3’ and 5’-TCCTGGCAAGACAGTGCAAT-3’ ; and for Gapdh, the primers were 5’-GGCACAGTCAAGGCTGAGAATG-3’ and 5’-ATGGTGGTGAAGACGCCAGTA-3’ . Assays were performed using KOD One® PCR Master Mix-Blue (Toyobo), as follows: 30 cycles at 98°C for 10 s, 60°C for 5 s, and 68°C for 3 s.
5
Molecular Detection of ESBL Genes
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All positive phenotypic ESBLs were tested using PCR amplification to identify the presence of blaTEM, blaSHV, blaOXA, blaCTX-M, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9, and blaCTX-M-25. DNA was extracted using NucleoSpin® (Düren, Germany) according to the manufacturer’s instructions. The PCR reactions were amplified with specific primers and optimized annealing temperature for each primer (Table-1 ) [20 , 21 (link)] through KOD One™ PCR Master Mix Blue (Toyobo, Japan) containing KOD DNA polymerase.
6
Soybean Transcriptomic Analysis Protocol
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Soybean genomic DNA was extracted by the CTAB method (Porebski et al., 1997 ). RNA was extracted from three replicate samples of soybean roots, stems, leaves, flowers, pods, and grains at four developmental stages (EM, MM, LM, and DS) using the TRIzol reagent (Invitrogen). RNA was reverse transcribed using HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). KOD One PCR Master Mix‐Blue (Toyobo, Osaka, Japan) was used for fragment amplification, and 2× Rapid Taq Master Mix (Vazyme) was used for PCR. qRT‐PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme) on a LightCycler 480 II instrument (Roche, Basel, Switzerland) (Chen et al., 2013 ). Soybean GmActin was used as the reference gene, and primers for PCR and qRT‐PCR are listed in Table S13 .
7
Optimized PCR Amplification Protocol
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KOD One™ PCR Master Mix ‐Blue (TOYOBO, KMM‐201) was used for PCR. The sequences of the primer pair were listed in Table S2 . The PCR protocol was: 95℃/1 minutes (1 cycle), 94℃/30 seconds, 70℃/45 seconds (2 cycles), 94℃/30 seconds, 68℃/45 seconds (5 cycles), 94℃/20 seconds, 66℃/1 minutes (29 cycles), and 4℃ hold.
8
Amplification and Sequencing of cyp51A Gene
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The promoter region (approximately 500 bp) of the cyp51A gene was amplified by colony PCR using a set of primers Pcyp51A-F(-500) and Pcyp51A-R (Supplemental Fig. S1 and Table S1). GoTaq Green Master Mix (Promega, Madison, WI, USA) or KOD One PCR Master Mix Blue (Toyobo, Osaka, Japan) was used for amplification. The amplified targeted fragments were purified and sequenced by the Sanger method, using either of the above primers. For full-length sequencing the cyp51A gene, a set of primers Pcyp51A-F(-500) and cyp51A-R were used to amplify the ORF, and cyp51A-SQ1, cyp51A-SQ2, and cyp51A-SQ3 were used for sequencing (Supplemental Table S1). The reference sequence was retrieved from the A. fumigatus A1163 genome.
9
Cloning of hACE2 into piggyBac Plasmid
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The hACE2 gene was cloned into the piggyBac plasmid (Systembiosciences, PB514B-2). Briefly, total RNA was extracted from 293T cells and reverse-transcribed using the SuperScript™ III First-Strand Synthesis System for RT-PCR (Invitrogen, Cat# 11904018). Then, hACE2 complementary DNA (cDNA) was amplified using the KOD One PCR Master Mix -Blue- (TOYOBO, Cat# KMM-201). The obtained fragment was digested with XbaI (NEB, Cat# R0145) and NotI (NEB, Cat# R0189) and ligated with PB514B-2, which was also digested with XbaI and NotI, using the TAKARA Ligation kit ver.2.1 (TAKARA Bio, Cat# 6022).
10
Genomic DNA Extraction and PCR
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The extraction of gDNA from tail samples was performed using the Hot Shot method and the extraction of gDNA from blood samples was performed using High Pure PCR Template Preparation Kit (Roche, Switzerland). Polymerase chain reaction (PCR) was performed using KOD One™ PCR Master Mix -Blue- (TOYOBO, Japan). Primer sequences and PCR conditions are detailed in the S2 Table .
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