Routine DNA manipulations were performed as described by Sambrook et al. [21 ]. All restriction enzymes were purchased from Thermo Fisher Scientific and used according to the manufacturers’ recommendations. PCR amplifications were carried out using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). DNA from agarose gels and PCR products were purified with the illustra™ GFX™ PCR DNA and Gel Band Purification kit (GE Healthcare Life Sciences). All DNA ligations were performed using T4 DNA Ligase (Thermo Fisher Scientific). DNA phosphorylation was performed using T4 Polynucleotide Kinase (Thermo Fisher Scientific) and DNA dephosphorylation with FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific). Plasmids were purified using the NZYMiniprep kit (NZYTech). DNA sequencing was performed with the ABI PRISM BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems). The sequencing reaction was purified by gel filtration and resolved in an ABI 3730XL sequencer.
Nzyminiprep kit
Manufactured by NZYTech
Sourced in Portugal
The NZYMiniprep kit is a DNA plasmid purification tool designed for the rapid and efficient isolation of plasmid DNA from bacterial cultures. The kit utilizes a silica-membrane-based technology to provide a simple and reliable method for the purification of high-quality plasmid DNA suitable for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (4 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (3 mentions)
Fastap thermosensitive alkaline phosphatase, by Thermo Fisher Scientific (2 mentions)
Pgem t easy vector, by Promega (2 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (2 mentions)
Abi prism bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Illustra gfx pcr dna and gel band purification kit, by GE Healthcare (1 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (1 mentions)
Purelink quick gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Gel doc 2000, by Bio-Rad (1 mentions)
Ez dna methylation lightning kit, by Zymo Research (1 mentions)
Quick gdna miniprep kit, by Zymo Research (1 mentions)
T4 ligase, by Thermo Fisher Scientific (1 mentions)
E gel electrophoresis system, by Thermo Fisher Scientific (1 mentions)
Taq dna polymerase, by Avantor (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Nucleobond bac 100 plasmid purification kit, by Macherey-Nagel (1 mentions)
Biophotometer, by Eppendorf (1 mentions)
Nzygelpure kit, by NZYTech (1 mentions)
19 protocols using nzyminiprep kit
1
Routine DNA manipulations protocol
2
Cloning and Sequencing of TdHSP101C Gene
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DNA was isolated from fresh young leaves using Citogene® DNA Purification Kit (Citomed). PCR amplification of TdHSP101C coding sequence targeting the protein C-terminal region (including the AAA+ and ClpB_D2-small conserved domains) was performed using primers previously designed by [21 ]. PCR reactions with 50 μl were prepared with: 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.25 mM dNTP’s, 1 mM each primer (forward 5’-GTTGGACAGTATGAGGCCGT-3’ ; reverse 5’-CATTTCACCCCCAATTCAACAG-3’ ), 0.5 U Taq polymerase and 25 ng DNA template. The following program was used: 3 min at 95°C; 30 cycles of 30 s at 95°C, 30 s at 60°C, 45 s at 72°C; termination by 5 min of final extension at 72°C. PCR products were separated through electrophoresis in 1.7% agarose gels stained with ethidium bromide and photographed using Bio-Rad GEL DOC 2000. Selected bands were gel isolated and purified using PureLink® Quick Gel Extraction Kit (Invitrogen) and cloned using TA Cloning® Kit (Invitrogen). Selected colonies were grown overnight in 5 ml LB broth containing 100 μg/ml ampicillin, plasmids were isolated using NZYMiniprep® kit (Nzytech) and finally sequenced through Sanger Sequencing.
3
Bisulfite Sequencing of Treg and Tcon
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Genomic DNA (gDNA) from Treg and Tcon was isolated with the Quick‐gDNA MiniPrep kit (Zymo Research). Bisulphite (BS) conversion of gDNA from all cell populations was performed at the same time with EZ DNA methylation lightning kit (Zymo Research).
TSDR and CAMTA1 gene regions were amplified with nonmethylation‐, BS DNA‐, strand‐specific forward (Fw) and reverse (Rev) primers and cloned into pGEM‐T Easy vector (Promega) via NcoI and NsiI restriction sites, as previously described.15 Plasmid DNAs from 22 to 24 clones were isolated using plasmid NZYMiniprep kit (NZYtech) and 20 to 22 positive clones were sequenced using reverse SP6 primer: 5′‐GTGACACTATAGAATACTC‐3′ (Stabvida). Sequences (AB1 files containing chromatograms) were aligned to each gene region's reference sequence using SeqMan software (DNA Star Lasergene 8). All nonmethylated cytosines (C) were identified by the presence of a thymidine (T) in BS‐converted sequences, whereas methylated C (mC) were identified by a C. Efficiency of BS conversion was confirmed by conversion of non‐CpG C to T. The percentage of methylation in each CpG position was determined by defining the proportion of mC in the total of 20. As both gene regions were amplified from the same BS DNA template, the methylation differences reflect the average methylation status of the cell population.
TSDR and CAMTA1 gene regions were amplified with nonmethylation‐, BS DNA‐, strand‐specific forward (Fw) and reverse (Rev) primers and cloned into pGEM‐T Easy vector (Promega) via NcoI and NsiI restriction sites, as previously described.
4
RAPD Amplification Product Isolation
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A RAPD amplification product present on M. luci and absent on M. ethiopica was purified from the agarose gel using the NucleoSpin® Gel and PCR Clean-up kit (Macherey Nagel), ligated into a pGEM®-T Easy Vector (Promega) and transformed in Escherichia coli JM109 High Efficiency Competent Cells (Promega), following the manufacturer’s instructions. Plasmid DNA was extracted from E. coli cells using a NZYMini Prep kit (Nzytech), and two positive clones were selected and were fully sequenced in both strands in an Automatic Sequencer 3730xl under BigDyeTM terminator cycling conditions at the Macrogen Company (Spain).
5
DNA Cloning and Sequencing Techniques
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DNA manipulations were carried out as described previously by Sambrook et al.47 . Restriction enzymes, T4 ligase and FastAP Thermosensitive Alkaline Phosphatase used in DNA cloning were purchased from Thermo Fisher Scientific and were used in accordance with the manufacturer’s instructions. DNA ligations were performed using T4 DNA Ligase. For PCR reactions Phusion High-Fidelity DNA Polymerase, also from Thermo Fisher Scientific, was used. Clean-up of DNA from agarose gels and restriction and PCR reactions was performed with GFX Gel Band Purification kit (GE Healthcare Life Sciences). Plasmid DNA extraction and clean-up was done using the NZYMiniprep kit from NZYTech. Plasmid DNA and PCR product Sanger sequencing was performed at STAB VIDA (https://www.stabvida.com/ ). All primers used in this work are listed in Supplementary Table 4 .
6
Cloning and Expression of CbcL Periplasmic Domain
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DNA sequence of the gene cbcL (GSU0274, GenBank accession number AAR33608.1 ) was obtained from G. sulfurreducens PCA genome database from Kyoto Encyclopedia of Genes and Genomes database (Kanehisa et al., 2016 (link)). Residues 30-279 (CbcL periplasmic domain) were amplified from genomic DNA using Phusion DNA polymerase (Thermo Scientific) together with primers with restriction sites for NotI and HindIII enzymes. The resulting DNA fragment and vector pVA203 (Londer et al., 2002 (link); Pokkuluri et al., 2004a (link)) were digested, the E-gel® electrophoresis system (Invitrogen) was used to purify them, and T4 DNA ligase (Thermo Scientific) was used to ligate the DNA fragment to the vector. The plasmid was further modified by the addition of a C-terminal Strep-tag®. Plasmids were propagated in Escherichia coli DH5α cells and colony screenings performed by PCR using Taq DNA polymerase (VWR). Positive clones were cultured in liquid medium. The plasmids were then purified using the NZYMiniprep kit (NZYTech) and sequenced by STABVida (Caparica, Portugal). The resulting plasmid pVA203-CbcL-St codes for the signal peptide of the protein OmpA from E. coli followed by the periplasmic domain of CbcL and a C-terminal Strep-tag®.
7
Purification and Amplification of BACmid DNA
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BACmid DNA for PCR amplification and transfection was obtained from E. coli using the NucleoBond BAC 100 Plasmid purification kit (Macherey & Nagel, Düren, Germany) according to the manufacturer’s instructions. Plasmid maintenance and amplification were performed from E. coli cells carrying pKD46 or pKD4 plasmid using the NZYMiniprep Kit (Nzytech, Lisboa, Portugal). The concentration of purified plasmid DNA preparations was determined by using a spectrophotometer BioPhotometer (Eppendorf, Hamburgo, Alemania).
8
Plasmid Detection in merA Strains
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All merA positive strains were tested for the presence of plasmids using the method proposed by Kado and Liu (1981 (link)) using the TOL plasmid of Pseudomonas putida KT2440 as positive control and the NZYMiniprep kit of NZYTech according to the manufacturer’s instructions. The resulting DNA was run on 0.8% agarose gels in TAE buffer for 60–90 min at 100 V.
9
Chs3 Truncations Characterization
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The different Chs3 truncations used in this work were obtained through homologous recombination in the chs3Δ yeast mutant after its cotransformation with the linearized centromeric plasmid pRS315-Chs3-GFP and different synthetic fragments (Genewiz) containing specific fusions within the Chs3 sequence to delete specific regions. After selecting transformants, we obtained their genomic DNA (Cheng and Jiang, 2006 ), which was later introduced by electroporation into electrocompetent Escherichia coli DH10β cells. The pRS315 plasmids were recovered from the transformed bacteria; they potentially contained the specific truncations marked at the C-terminus with GFP and under the control of the endogenous promoter of Chs3. Plasmids were purified using the NZYMiniprep Kit (Nzytech) and sequenced to confirm the presence of the correct construct. ∆63Chs3 and Chs3∆37 truncations had been made previously (see Supplemental Table S3). All constructs used were tagged with GFP and were tested in a chs3∆ background to ensure that the assayed construct was the only copy of Chs3 in the cell.
10
Sanger Sequencing of Genetic Variations
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PCR products were purified using NZYGelpure kit (NZYTech, Lda.) following manufacturer’s instructions and sent to GATC-Biotech (Eurofins Scientific) for Sanger sequencing of both strands. Sequences were corrected for accurate base calling by comparison of both strands using CodonCode Aligner (CodonCode Corporation). Sequence alignments were performed with ClustalW83 (link) and manually edited with BioEdit84 . Sequences have been deposited in GenBank under accession numbers: MW288771–MW288823, MW284939–MW284941 and MW284942–MW284950.
The presence of double peaks throughout some of the sequence reads were taken as evidence for genetic variation in the sample. In these cases, the PCR products were cloned using CloneJET PCR Cloning Kit (ThermoFisher Scientific Inc.) and DH5α Escherichia coli competent cells (Invitrogen) under manufacturer’s conditions using half of the indicated volumes. Plasmids were isolated using the NZYMiniprep Kit (NZYtech) and 5 clones per locus were sent to GATC-Biotech for Sanger sequencing.
The presence of double peaks throughout some of the sequence reads were taken as evidence for genetic variation in the sample. In these cases, the PCR products were cloned using CloneJET PCR Cloning Kit (ThermoFisher Scientific Inc.) and DH5α Escherichia coli competent cells (Invitrogen) under manufacturer’s conditions using half of the indicated volumes. Plasmids were isolated using the NZYMiniprep Kit (NZYtech) and 5 clones per locus were sent to GATC-Biotech for Sanger sequencing.
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