To construct pBAD-rbsR for complementation assays, the rbsR gene was first amplified using oligonucleotide pair, oCML24 and oCML37. Oligonucleotides used for PCR amplification were purchased from Sigma Aldrich and are listed in Table 2 . The PCR product and plasmid pBAD30 were digested with KpnI (NEB) and HindIII (NEB) at 37°C for 2 h. The digested PCR product was subsequently ligated into compatibly digested pBAD30 using T4 DNA ligase (NEB) according to the manufacturer's instructions. Plasmid pQE80-rbsR was constructed using Oligonucleotides oCML24 and oCML36 to amplify the rbsR open reading frame (ORF). The amplified fragment was digested with KpnI (NEB) and HindIII (NEB) and ligated with compatibility digested pQE80-oriT. For the construction of pBAD-rbsK, Oligonucleotides oREM726 and oREM727 were used to amplify the rbsK ORF and the subsequent PCR product was digested with KpnI and HindIII. This was ligated with compatibly digested pBAD33 to form pBAD-rbsK. All plasmids were subjected to sequencing (GATC Biotech) to confirm the sequence was correct.
Hindiii
Manufactured by New England Biolabs
Sourced in United States, United Kingdom, China, Germany, Japan, Canada
HindIII is a type II restriction endonuclease enzyme that recognizes and cleaves the palindromic DNA sequence 5'-A^AGCTT-3'. It is widely used in molecular biology and genetic engineering applications for DNA manipulation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bamhi, by New England Biolabs (58 mentions)
Ecori, by New England Biolabs (44 mentions)
T4 dna ligase, by New England Biolabs (29 mentions)
Xhoi, by New England Biolabs (24 mentions)
Xbai, by New England Biolabs (24 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (10 mentions)
Ecorv, by New England Biolabs (9 mentions)
Biotin 14 dctp, by Thermo Fisher Scientific (8 mentions)
T4 dna ligase, by Thermo Fisher Scientific (7 mentions)
Pcr dig probe synthesis kit, by Roche (6 mentions)
T4 ligase, by New England Biolabs (6 mentions)
Bsrgi, by New England Biolabs (6 mentions)
Qiaquick gel extraction kit, by Qiagen (6 mentions)
Hindiii, by Thermo Fisher Scientific (6 mentions)
Dneasy blood tissue kit, by Qiagen (5 mentions)
Streptavidin beads, by Thermo Fisher Scientific (5 mentions)
Bglii, by New England Biolabs (5 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
Bamhi hf, by New England Biolabs (4 mentions)
Qx200 droplet generator, by Bio-Rad (4 mentions)
328 protocols using hindiii
1
Construction of rbsR and rbsK Cloning Vectors
2
Quantifying NHEJ in U2OS and Fibroblasts
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U2OS EJ5 reporter cell lines were seeded in triplicate for each condition and were left untreated, pre-treated media control, or pre-treated with 10μM purified full length 3E10. The next day, the I-SceI expression plasmid (4 μg) was transfected into 1 × 106 cells per replicate using the Amaxa Nucleofector (Lonza) 72 hours before analysis. Cells were analyzed for GFP expression by flow cytometry, and data were analyzed using the FlowJo software (Tree Star Inc.). Luciferase reporter DNA plasmids for NHEJ were digested with HindIII (New England Biolabs, Inc. #R3104) according to HindIII manufacturer instructions. Digestion was confirmed on a 0.7% agarose gel and then purified using the QIAquick PCR Purification Kit (QIAGEN # 28104). Primary human skin fibroblasts were seeded at 1 × 105 cells per well in a 12-well dish the day before transfection. Transfection steps were carried out as above. Cells were collected and lysed in 1× Passive Lysis Buffer (Promega) 24 h after transfection and analysis was continued as above. Each experiment contained technical triplicates per condition. Statistical significance was determined using the unpaired t-test (GraphPad Prism).
3
Optimized Hi-C Nuclei Sample Preparation
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Each sample of two million nuclei was resuspended in 500 μl of 1× NEB2.1 on ice. Next, 600 U of HindIII (NEB R0104T) was added to the sample in order to obtain 50 U HindIII/μg of DNA and then treated as described immediately below.
Next, control and pre-digestion samples were incubated at 37°C on a thermocycler (900 rpm for 30 sec every 4 min) for 5 min up to 16 h. Afterward, samples were placed on ice for 10 min. For DpnII-seq and assessment of fragmentation level, a final volume of 10 mM of EDTA was added to inactivate the endonuclease, followed immediately by the DpnII-seq protocol (details of protocols below. DpnII-Seq) or DNA purification for fragment analyzer analysis. For Hi-C, we proceeded immediately to the first step of the protocol (crosslinking as described below). For microscopy, nuclei samples were cross-linked with a 4% final concentration of paraformaldehyde.
Next, control and pre-digestion samples were incubated at 37°C on a thermocycler (900 rpm for 30 sec every 4 min) for 5 min up to 16 h. Afterward, samples were placed on ice for 10 min. For DpnII-seq and assessment of fragmentation level, a final volume of 10 mM of EDTA was added to inactivate the endonuclease, followed immediately by the DpnII-seq protocol (details of protocols below. DpnII-Seq) or DNA purification for fragment analyzer analysis. For Hi-C, we proceeded immediately to the first step of the protocol (crosslinking as described below). For microscopy, nuclei samples were cross-linked with a 4% final concentration of paraformaldehyde.
4
Cloning and Expressing laEBLN-1 Isoforms
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cDNAs reverse-transcribed from laEBLN-1v1 and laEBLN-1v2 mRNAs were amplified by PCR using Blend taq (TOYOBO) with the primer pairs listed in S5 Table . The PCR product of laEBLN-1v1 was ligated into 3×FLAG-CMV-14 vector (SIGMA) using Ligation high ver.2 (TOYOBO) after digestion with the restriction enzymes HindIII and BamHI (New England Biolabs) at 37°C for 60 min. The PCR product of laEBLN-1v2 was ligated into V5-His-TOPO vector (Life Technologies) according to manufacture’s instructions. The plasmids transformed into TOP10 Competent Cells (Life Technologies) were collected using PureYield Plasmid Midiprep System (Promega) after confirming the insert with nucleotide sequencing. In addition, sub-cloning of ORFs in laEBLN-1v1 and laEBLN-1v2 into pcDNA3.10 vector (Thermo Fisher Scientific) was conducted using the above plasmids as templates for PCR, which were digested with HindIII and BamHI (New England Biolabs).
5
Targeted Genome Editing Verification
6
Enzymatic Digestion and Gel Electrophoresis of λ-DNA
7
Optimized Hi-C Nuclei Sample Preparation
8
Cloning and Expression of Outer Membrane Proteins
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The ompA, ompC, ompD, and ompF genes were amplified by colony PCR with their respective cloning primers (Table 2 ) . The amplified PCR products and empty pQE60 vector were subjected to restriction digestion by specific restriction enzymes such as NcoI (NEB) and HindIII (NEB) for ompA, BamHI (NEB), and HindIII (NEB) for ompC, ompD, and ompF in the CutSmart buffer (NEB) at 37°C for 2–3 h. Double digested insert and vector were subjected to ligation by a T4 DNA ligase in 10X ligation buffer (NEB) overnight at 16°C. The ligated products and the empty vector were transformed into the bacteria to generate complemented, over-expression, and empty vector strains. Complementation and over-expression were initially confirmed by restriction digestion of recombinant plasmid. The expression level of ompA in the knockout, complemented, and empty vector strains were further confirmed by RT-qPCR using ompA expression primer (Table 2 ) .
9
Plasmid DNA Substrate Generation
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Plasmid DNA substrates were generated either by cloning oligoduplexes assembled after annealing complementary oligonucleotides containing PAM and protospacer sequences into pUC19 plasmid over EcoRI (New England Biolabs) and HindIII (New England Biolabs) restriction sites or by cloning PCR products containing PAM and protospacer sequences via blunt end ligation over end repaired EcoRI and HindIII restriction sites.
Fluorescently labeled DNA substrates were generated by annealing partially complementary oligonucleotides containing PAM and protospacer sequences and PCR amplifying them with primers containing 5′‐6‐FAM (nontarget strand) or 5′‐6‐ROX (target strand) (IDT) dyes.
The sequences of the DNA substrates and links to plasmid sequences are provided in the DatasetEV1 .
Fluorescently labeled DNA substrates were generated by annealing partially complementary oligonucleotides containing PAM and protospacer sequences and PCR amplifying them with primers containing 5′‐6‐FAM (nontarget strand) or 5′‐6‐ROX (target strand) (IDT) dyes.
The sequences of the DNA substrates and links to plasmid sequences are provided in the Dataset
10
Gene Copy Number Identification in Cajanus spp.
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In order to identify the gene copy number of F3′5′H_2 in C. cajan and C. platycarpus, high-quality genomic DNA was isolated from young leaves of plants according to Cetyl trimethyl ammonium bromide (CTAB; Millipore Sigma, Burlington, MA, USA) method [50 (link)]. Approximately 15 μg purified genomic DNA from both the Cajanus spp. was digested overnight separately with BamHI and HindIII (New England Biolabs, Ipswich, MA, USA). Additionally, about 300 pg of pCambia 2300 vector consisting of CpF3′5′H_2 gene was linearized with HindIII (New England Biolabs, Ipswich, MA, USA) and used as the positive control. Overnight-restricted DNA samples were electrophoretically separated on a 0.8% agarose gel in Tris-acetate EDTA (TAE; Millipore Sigma) buffer. The separated fragments were further blotted onto a positively charged nylon membrane (Millipore Sigma) and hybridized with a Digoxigenin (DIG)-labelled 543-bp F3′5′H_2 probe. Membrane washing and the development of X-ray film were carried out according to the manufacturer’s instructions (Roche Holding AG, Basel, CH).
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