Isopropyl-β-D-thiogalactopyranoside (IPTG), anhydrous sodium phosphate, and dibasic potassium phosphate were purchased from DOT Scientific. The buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glycerol, and imidazole were purchased from Fisher Scientific. Kanamycin was purchased from Teknova. Magnesium sulfate heptahydrate, L-β-homolysine and D-Lysine were purchased from Chem-Impex International. Ni(II)-nitrilotriacetic acid agarose (Ni-NTA) resin was purchased from Qiagen. L-Allylglycine was purchased from Cayman Chemical Company. 4,4,5,5-[2H2]-L-Lysine (d4-L-Lys) was purchased from Cambridge Isotope Laboratories. L-norleucine was purchased from Alfa Aesar. Trans-4,5-dehydro-DL-Lysine was purchased from Bachem. 6-Hydroxy-L-norleucine was purchased from Acrotein Chembio Inc. 4RS-Cl-DL-Lysine was purchased from Akos GmbH (Steinen, Germany). EZNA Plasmid DNA Mini Kit was purchased from Omega Bio-Tek. Oligonucleotide primers were purchased from Integrated DNA Technologies. Polymerase chain reaction (PCR) reagents, Q5 DNA Polymerase Master Mix, restriction enzymes, restriction enzyme buffers, and T4 DNA ligase were purchased from New England Biolabs. Escherichia coli (Ec) DH5α and Rosetta(DE3) competent cells were purchased from Novagen. 57Fe0 was purchased from ISOFLEX, USA. All other chemicals were purchased from Millipore Sigma.
Ezna plasmid dna mini kit
Manufactured by Omega Bio-Tek
Sourced in United States
The EZNA Plasmid DNA Mini Kit is a laboratory product designed for the rapid and efficient isolation of plasmid DNA from bacterial cultures. It utilizes a silica-based membrane technology to purify plasmid DNA from small-scale samples.
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Lab products found in correlation
E z n a cycle pure kit, by Omega Bio-Tek (4 mentions)
Pselect gfpzeo, by InvivoGen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Kanamycin, by Teknova (1 mentions)
Imidazole, by Thermo Fisher Scientific (1 mentions)
Ni 2 nitrilotriacetic acid agarose ni nta resin, by Qiagen (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Oligonucleotide primers, by Integrated DNA Technologies (1 mentions)
Taq dna polymerase, by New England Biolabs (1 mentions)
E coli bl21 gold de3 cells, by Agilent Technologies (1 mentions)
Ta cloning kit, by Thermo Fisher Scientific (1 mentions)
E z n a total rna kit, by Omega Bio-Tek (1 mentions)
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Gotaq polymerase, by Promega (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Gelred, by Biotium (1 mentions)
Gel doc system, by Bio-Rad (1 mentions)
Purelink genomic dna mini kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using ezna plasmid dna mini kit
1
Protein Expression and Purification Protocol
2
Random Mutagenesis of Fluorescent Proteins
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Three rounds of random mutagenesis, starting from the mSandy1 gene and then on the pooled RFP genes from the top 1% cells obtained after cell sorting (see below), were performed using the protocol described by McCullum et al.46 (link) Briefly, four cycles of error-prone polymerase chain reaction using Taq DNA polymerase (5 U) in 1× standard Taq buffer pH 8.3 (New England Biolabs) supplemented with MnCl2 (0.5 mM) and a mixture of deoxynucleotides (1 mM dTTP, 1 mM dCTP, 0.2 mM dATP, and 0.2 mM dGTP), were used to introduce approximately two mutations on the 732-bp RFP open reading frame. The resulting mutant library was cloned into pET-11a (Novagen) via NdeI and BamHI. Plasmids were transformed into E. cloni 10G Elite electrocompetent cells (Lucigen) and plated onto lysogeny broth (LB) agar supplemented with 100 μg mL−1 ampicillin. Following incubation at 37 °C overnight, all colonies on the agar plate were collected using 1 mL of LB supplemented with 100 μg mL−1 ampicillin, and the plasmid pool was harvested by DNA extraction (E.Z.N.A. Plasmid DNA Mini Kit, Omega Bio-tek). Purified plasmid DNA was then transformed into electrocompetent E. coli BL21-Gold (DE3) cells (Agilent) for protein expression.
3
Construction and Validation of pCRISPR-SPC1 Plasmid
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The pcrispr-spc1 plasmid which contains a single repeat and spc1 was constructed using a two-piece Gibson assembly with primers noted in S1 Table . Briefly, the repeat-spc1 region was amplified using primers F063/A010 from a plasmid containing the wild-type repeat-spacer array, and primers F062/L162 were used to amplify the backbone from a pcrispr-cas plasmid lacking all repeats and spacers [27 (link)]. PCR products were then purified with EZNA Cycle Pure Kit (Omega Bio-tek) and Gibson assembled. The assembled construct was introduced into S. aureus RN4220 via electroporation, and transformants were subjected to PCR and DNA sequencing using primers A200/F111 to confirm the presence of a single repeat and spacer. The confirmed construct was then purified with EZNA Plasmid DNA Mini Kit (Omega Bio-tek) and introduced into S. epidermidis LM1680 strains via electroporation.
4
Cloning and Validation of HIV-1 Nef and Vpu Constructs
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Nef and vpu amplicons were cloned into eukaryotic expression vectors as described (28 (link), 30 (link)). Briefly, second-round products were purified using the E.Z.N.A Cycle Pure kit (Omega Bio-tek). Each nef or vpu amplicon was then cloned into a modified version of pSELECT-GFPzeo (In vivoGen) that contained 5’ AscI and 3’ SacII sites, where expression is driven by a composite hEF1-HTLV promoter. pSELECT-GFPzeo also features an independent CMV/HTLV promoter driving the expression of GFP. The pSELECT-GFPzeo vector used for vpu cloning was further modified to include the HIV-1 Rev Responsive Element (RRE) sequence downstream of the multiple cloning site to enhance Vpu expression (pSELECT-RRE-GFP). Following ligation, DNA products were transformed into E. cloni 10G chemically competent cells (Lucigen) and plated on Luria-Bertani (LB) agar containing Zeocin. Colonies were isolated, grown in LB medium containing Zeocin, and plasmid DNA was purified using the E.Z.N.A Plasmid DNA Mini kit (Omega Bio-tek). Plasmid clones were validated by Sanger sequencing. Following phylogenetic authentication (see below), one intact nef and one intact vpu clone per participant was selected for in vitro functional analysis (Supplementary Figure 1 ).
5
Hybridoma RNA Extraction and Sequence Analysis
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RNA was isolated from expanded hybridoma cells using the EZNA total RNA kit (Omega BioTek) according to the manufacturer’s protocol. cDNA was obtained using the Superscript IV reverse transcriptase kit. Following this, PCR was conducted in two steps using established primers for the heavy chain and kappa and lambda light chains (85 (link)). Samples were analyzed by agarose gel electrophoresis and purified PCR products (EZNA Cycle Pure kit; Omega Bio-Tek) were cloned into the pCR2.1 vector using the original TA Cloning kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Plasmids were purified from positive DH5α colonies with an EZNA plasmid DNA minikit (Omega Bio-Tek) and submitted to Genewiz for sequencing. Sequences were analyzed using IMGT/V-Quest (86 (link)).
6
Phh-grd and Phh-lcch3 Cloning and Transcription
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The full-length Phh-grd variants and Phh-lcch3 were amplified using GoTaq polymerase (Promega) and cloned in the pTB207 expression vector using the In-Fusion HD Cloning Kit (Takara Bio Europe SAS, Saint-Germain-en-Laye, France) as described (Lamassiaude et al., 2021 (link)). Recombinant plasmids were purified using E.Z.N.A. Plasmid DNA Mini Kit (Omega Bio-Tek, Inc., Norcross, GA), and correct cloning was confirmed by sequencing (Eurofins Genomics). cRNAs were obtained from plasmids linearized by Msc1 (Thermo Fisher Scientific) using mMessage mMachine T7 transcription kit (Thermo Fisher Scientific) following the manufacturer's instructions. cRNA concentrations were measured by spectrophotometry (NanoDrop; Thermo Fisher Scientific), and integrity of cRNAs was confirmed by running 500 ng in 1% agarose gel in TAE buffer (40 mM Tris-acetate–1 mM EDTA).
7
Cloning and Validation of HIV-1 Nef and Vpu Constructs
8
Plasmid DNA Degradation Analysis in E. coli
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The plasmid DNA degradation analysis in E. coli cells was carried out as described previously (Ma et al., 2014 (link)). In brief, E. coli BL21(DE3) with recombinant or empty pBAD 33.1 have grown in LB containing chloramphenicol to an OD600 of 0.6–0.8. The expression of putative effector alone (or with cognate immunity) was then induced by 0.2% L-arabinose for 3 h. Equivalents of 1 ml of cells at OD600 = 1.0 from each culture were pelleted for plasmid extraction. Plasmid DNA was extracted using an E.Z.N.A. Plasmid DNA Mini Kit (Omega Bio-Tek, Inc., GA, United States). Plasmid DNA samples were analyzed by gel electrophoresis on a 1% agarose gel containing 1× GelRed (Biotium, Hayward, CA, United States) and run at 10 V/cm for 30 min. DNA was visualized under UV light transillumination using a Gel Doc system (BioRad, La Jolla, CA, United States).
9
Plasmid and Genomic DNA Extraction
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Plasmid DNA was routinely extracted using the EZNA Plasmid DNA Mini Kit (Omega Bio-tek) and eluted in 10 mM Tris-HCl pH 8.5. Genomic DNA was isolated from Burkholderia with the PureLink Genomic DNA Mini Kit (Invitrogen) or by standard isopropanol precipitation and solubilized in 10 mM Tris-HCl pH 8.5.
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