An approximately 450-bp DNA fragment of the putative bisucaberin B (1 ) biosynthetic gene cluster was PCR amplified from T. mesophilum genomic DNA using degenerate primers. The primers (forward: 5′-GTNGCNAAYAAYGGNCGYATYGGGTT-3′, reverse: 5′-SWNARNCCRCGCATRAAACCCATRTT-3′) were designed to amplify a sequence conserved among the genes encoding the known amide-bond formation enzymes (enzymes D) of the HSD-based siderophore clusters (see Supplementary Materials Sequences S3–S9 ). The PCR program consisted of an initial denaturation at 95 °C for 2 min, followed by 30 cycles at 94 °C for 20 s, 50 °C for 20 s, and 72 °C for 70 s. Amplified DNA was cloned into T-vector pMD20 (Takara), and then transformed into NEB 10-beta competent cells (New England Biolabs, Ipswich, MA, USA). Cloned DNA was subjected to DNA sequencing.
Neb 10 beta competent cells
Manufactured by New England Biolabs
Sourced in United States
NEB 10-beta competent cells are a strain of Escherichia coli that have been engineered to be chemically competent for DNA transformation. They are suitable for cloning and plasmid propagation.
Automatically generated - may contain errors
Lab products found in correlation
Kld enzyme mix, by New England Biolabs (2 mentions)
Q5 hot start high fidelity dna polymerase, by New England Biolabs (2 mentions)
User cloning, by New England Biolabs (2 mentions)
T vector pmd20, by Takara Bio (1 mentions)
Monarch miniprep kits, by New England Biolabs (1 mentions)
Neb pcr cloning kit, by New England Biolabs (1 mentions)
Q5u dna polymerase, by New England Biolabs (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Gel pcr dna fragments extraction kit, by IBI Scientific (1 mentions)
5 protocols using neb 10 beta competent cells
1
Amplification of Putative Bisucaberin B Biosynthetic Gene
2
Precise APOBEC1 Variant Generation
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PCR was performed using Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). Plasmids for BE and sgRNA were constructed using USER cloning (New England Biolabs) from previously reported plasmids1 (link). DNA vector amplification was carried out using NEB 10beta competent cells (New England Biolabs). Site-directed mutagenesis of APOBEC1 variants was done using blunt-end ligation. Briefly, a primer with an overhang containing the desired point mutation was used to amplify the appropriate vector plasmid by PCR. KLD enzyme mix (New England Biolabs) was used to phosphorylate and circularize the PCR product prior to transformation.
3
Precise APOBEC1 Variant Generation
4
Plasmid Propagation and Bacmid Generation
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Table 1 outlines the plasmids used in this study. Plasmids were propagated using NEB-10-beta competent cells (NEB) and purified using Monarch miniprep kits (NEB). Bacmids were generated using the Multibac system [27 (link)] and purified using alkaline lysis method followed by isopropanol precipitation and resuspension in TE buffer.
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5
Bisulfite Sequencing of Genomic DNA
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Genomic DNA was extracted from mouse (Mus musculus) or rat (Rattus norvegicus) lung and testis tissue by phenol-chloroform extraction followed by ethanol precipitation. Human lung and testis genomic DNA from normal adult tissue was purchased (BioChain). The EZ DNA Methylation-Gold Kit (Zymo Research) was used to treat 1000 ng of genomic DNA according to manufacturer instructions. The primers that were used to amplify the bisulfite treated genomic DNA were designed using the MethPrimer software (Li and Dahiya, 2002 (link)) (Table 1 ). The DNA fragments were PCR amplified with the Q5U DNA polymerase (New England BioLabs) and then gel purified using the Gel/PCR DNA Fragments Extraction Kit (IBI Scientific). The gel purified DNA fragments were cloned into the pMiniT 2.0 vector using the NEB PCR Cloning Kit (New England BioLabs) and then transformed into NEB 10-beta competent cells (New England BioLabs). Plasmid DNA was extracted from ten positive clones using the GeneJET Plasmid Miniprep Kit (Thermo Scientific) and Sanger sequenced (EuroFins Genomics). The methylation status of these individual CpG clones were determined with the Quantification Tool for Methylation Analysis (QUMA) software (Kumaki et al., 2008 (link)).
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