To confirm the role of tmexCD1-toprJ1 in tigecycline resistance, a 7,205 bp full-length ORF of tmexCD1-toprJ1 was amplified by PCR (Table S1). Subsequently, 0·3 pmol of purified PCR product was incubated with 1 μL T-Vector pMD-19 (TaKaRa) and 5 μL DNA Ligation Mighty Mix (TaKaRa) at 16°C overnight. The recombinant plasmid pUC19-tmexCD1-toprJ1 was then transformed into competent E. coli DH5α by incubating on ice for 30 min followed by incubating for 45 s at 42°C. Immediately thereafter, the DH5α was transferred to SOC medium (TaKaRa) at 37°C for one hour and then was coated on Luria–Bertani Agar (LB) medium containing 100 μg/ml of ampicillin at 37°C overnight. Visible strains after overnight were verified by PCR and then subjected to susceptibility testing using broth microdilution method. The recombinant vector was then extracted and transferred into K. pneumoniae ATCC 13883 via electroporation.
Dna ligation mighty mix
Manufactured by Takara Bio
Sourced in United States
DNA Ligation Mighty Mix is a high-efficiency DNA ligation reagent designed for the rapid and reliable ligation of DNA fragments. It contains a proprietary blend of enzymes and buffers optimized for ligation reactions.
Automatically generated - may contain errors
Lab products found in correlation
Soc medium, by Takara Bio (1 mentions)
Pmd19 t vector, by Takara Bio (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Takara taq polymerase, by Takara Bio (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Pgem t vector, by Promega (1 mentions)
2 protocols using dna ligation mighty mix
1
Evaluating tmexCD1-toprJ1 Role in Tigecycline Resistance
2
Profiling DNA Methylation in Myeloid Cells
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Extracted DNA from sorted myeloid cells was subjected to bisulphite treatment via EZ DNA Methylation Gold Kit (Zymo Research, California, USA), followed by PCR amplification using TaKaRa Taq polymerase (TaKaRa Bio Inc., Shiga Prefecture, Japan) for PD-L1, TIM-3, galectin-9, VISTA, ARG1, MMP9 and TGF-β1. The PCR products were cloned into pGEM-T-vector (Promega Corporation, Wisconsin, USA) using DNA Ligation Mighty Mix (TaKaRa Bio). Methylation primers were designed by MethPrimer database (https://www.urogene.org/methprimer/index1.html ). Sequences are listed in Supplementary Table 1b. Seven colonies were picked from each sample and plasmid extraction was performed using GENEJET Plasmid miniprep Kit (Thermo Fisher Scientific). M13 reverse primer was used for Sanger sequencing (Supplementary Table 1c).
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