All chemicals were reagent-grade or better and purchased from Sigma-Aldrich (Steinheim, Germany) unless otherwise stated. 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was obtained from Amresco (Solon, OH, USA). Restriction enzymes, ligase and standards for Agarose gel electrophoresis (GeneRuler DNA Ladder Mix) were obtained from Fermentas (Vilnius, Lithuania) while the Phusion polymerase was from New England BioLabs (Ipswich, UK). SDS-PAGE protein standard (Precision Plus Protein prestained standard) was from BioRad (Herts, UK). The cloning vector pJET 1.2 was purchased from Fermentas and the expression vector pET21a from Novagen (Madison, WI, USA). The expression vectors pPICZB and pPICZαA, E. coli strain BL21 (DE3) and P. pastoris strain X-33 were purchased from Invitrogen (Carlsbad, CA, USA). E. coli NEB 5-alpha was from New England BioLabs. The HisPrep FF 16/10 column was from GE Healthcare Bioscience AB (Uppsala, Sweden). F. oxysporum strain G12 was kindly provided by Gerhard Adam (Department of Applied Genetics and Cell Biology, BOKU Vienna, Austria).
Generuler dna ladder mix
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Lithuania
The GeneRuler DNA Ladder Mix is a ready-to-use solution of DNA fragments of known sizes, designed for estimating the size of DNA molecules in agarose gel electrophoresis. It provides a molecular weight reference for the separation of DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Phusion polymerase, by New England Biolabs (2 mentions)
Qiaquick gel extraction kit, by Qiagen (2 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (2 mentions)
Gotaq g2 flexi dna polymerase, by Promega (2 mentions)
C1000 touch thermal cycler, by Bio-Rad (2 mentions)
Molecular imager gel doc xr, by Bio-Rad (2 mentions)
Dneasy kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Ppiczαa, by Thermo Fisher Scientific (1 mentions)
Ppicz b, by Thermo Fisher Scientific (1 mentions)
Pjet1, by Thermo Fisher Scientific (1 mentions)
E coli bl21 de3 strain, by Thermo Fisher Scientific (1 mentions)
P pastoris strain x 33, by Thermo Fisher Scientific (1 mentions)
E coli neb 5 alpha, by New England Biolabs (1 mentions)
Hisprep ff 16 10 column, by GE Healthcare (1 mentions)
Dna loading buffer, by Thermo Fisher Scientific (1 mentions)
Chemocam imager 3, by Intas (1 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2h tetrazolium bromide mtt, by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
32 protocols using generuler dna ladder mix
1
Heterologous Expression of Fungal Enzyme
2
ITS1 PCR Amplification and Digestion
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The samples were analyzed for ITS1 PCR using 400 nM primers: LITSR: 5′-CTTG GATCATTTTCCGATG-3′ and L5.8S 5′-TGA TAC CAC TTA TCG CAT T-3′ [12 (link)]. The reaction was carried out with the PCR-Ready Supreme mix (Syntezza Bioscience, Jerusalem, Israel) in 25 μL of total reaction. Amplification conditions were as described previously [12 (link)]. PCR products (8–15 μL) were digested with Hae III enzyme, according to the manufacturer's instructions. The amplicons of about 300–350 bp were analyzed on 1.5% agarose gels and the restriction fragments on 4% agarose gels by electrophoresis at 100 V in 1X Tris-acetate-EDTA buffer (0.04 M Tris acetate and 1 mM EDTA, pH 8) and visualized by UV light after being stained with ethidium bromide (0.3 μg/mL). The GeneRuler DNA ladder Mix (Fermentas, MBI) was used as the DNA molecular marker.
3
Agarose Gel Electrophoresis Protocol
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A 2% gel was prepared by dissolving agarose powder in 1x TAE Buffer (50x: 50 mM EDTA, 1 M acetic acid, pH 8.0, Carl Roth, Karlsruhe, Germany) and microwave heating for about 1 minute. The solution was then cooled to approximately 55–60°C and ethidium bromide (EtBr) was added (0.1 μl / ml of 10 mg/ml stock solution, Sigma Aldrich, Steinheim Germany), 1x TAE buffer was used for electrophoresis; the DNA samples were diluted into DNA loading-buffer (Fermentas, St. Leon-Rot, Germany), a current between 1–10 Volts/cm was applied. The DNA was visualized by UV using the CHEMOCAM Imager 3.2 (INTAS, Göttingen, Germany) and the size of the DNA was determined using the GeneRuler DNA Ladder Mix (Fermentas, St. Leon-Rot, Germany). In order to analyse the concentration of the amplicons by band intensity, the ImageJ software (NIH, Bethesda, MD, USA) was used.
4
Preparation and Characterization of DOTAP Lipoplexes
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1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP) was purchased from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol (Chol) was obtained from Shanghai Bio Life Science and Technology Co. Ltd. (Shanghai, China). Methoxy poly (ethylene glycol)-succinyl-cholesterol conjugate (mPEG-suc-Chol) and folate-poly (ethylene glycol)-succinyl-cholesterol conjugate (F-PEG-suc-Chol) were synthesized as previously described [19 (link), 43 (link), 44 (link)]. A DNA ladder, Gene Ruler DNA ladder mix (SM0331) and loading buffer were obtained from Fermentas (Thermo Fisher Scientific Inc., Waltham, MA, US). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich, Co. (St. Louis, MO, US). DNase I was purchased from Roche diagnostics GmbH (Roche Applied Science Mannheim, Germany). Triton X-100 was obtained from Sanland Chemical Co., Ltd. (Los Angeles, CA, US). Anti-FOLR1 antibody (LSB5727) was provided from LifeSpan Biosciences (Seattle, WA, US). All the reagents used in this study were analytical grade (AR).
5
Molecular Characterization of Transgenic Plants
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DNA was isolated from putative transgenic plants, positive and negative controls using a standard kit NeoPrep100 DNA plant (Neogene Therapeutics, Inc., City (?), Ukraine) according to the manufacturer’s instructions. The transformation events were confirmed by standard PCR techniques as by Sambrook with PCR MIX 2-R kit (Neogene Therapeutics, Inc.) (Sambrook et al., 1989). The primers and expected size of the PCR fragments are shown in the Table. Gene Ruler DNA Ladder Mix (Fermentas Inc., Waltham, MA, USA) was used as molecular weight markers.
6
Deletion of Salmonella virulence genes
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DNA manipulations and isolation of chromosomal and plasmid DNA were performed according to standard protocols [53 ], and following the manufacturersʼ instructions. GeneRuler™ DNA Ladder Mix (Fermentas, St. Leon-Rot, Germany) was used as a marker for DNA analysis. Plasmid DNA was transformed via electroporation using a Bio-Rad Gene pulser II as recommended by the manufacturer and as described previously [54 (link)]. Polymerase chain reactions (PCRs) were carried out with Taq polymerase (Fermentas). As template for PCR, chromosomal DNA, plasmid DNA, or an aliquot of a single colony resuspended in 100 μl H2O was used. Oligonucleotides used in this study are listed in Additional file 7 . Genes invA, STM3254 and gatR-HTH were deleted using the λ-Red recombinase [32 (link)]. Briefly, PCR products containing the kanamycin resistance cassette of plasmid pKD4 and the flanking FRT sites were generated using primers of 70 nucleotides in length that included 20 nucleotides priming sequences for pKD4 as template DNA. The fragments were transformed into ST4/74 cells harboring plasmid pKD46, and the allelic replacement of the target genes was controlled by PCR. A nonpolar deletion mutant was obtained by transformation with pCP20 and validated by PCR analysis and DNA sequencing.
7
Molecular Profiling of Microbial Biogenic Amines
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The presence of the tyrosine decarboxylase gene tdcA, the histidine decarboxylase gene hdcA, the ornithine decarboxylase gene odc, and the aguA and aguD genes from the agmatine deiminase cluster (AgdI), was checked by PCR using the primer pairs P2for and P1-rev [33] , JV16HC and JV17HC [34] , ODC3 and ODC16 [35] , and Seq1 and Seq2 [17] , respectively. The PCR conditions were those described in [33] [34] [35] 17] , respectively and were performed in a MyCyclerTM thermal cycler (Bio-Rad, Spain) using DreamTaq polymerase (Fermentas, Lithuania). Total DNA from the strains was obtained as previously described [36] and used as a template in PCR. Total DNA from the tyramine-and putrescine-producing strain Enterococcus faecalis V583 [27] , from the ODC+ strain Lactobacillus saerimneri 30A [37] , and from the histamine-producer Lactobacillus buchneri B301 [38] , were used to provide positive controls.
PCR products were separated in 0.8% (w/v) agarose gels in 1XTAE buffer, and visualized after staining with ethidium bromide using a GelDoc 2000 system (Bio-Rad, Hercules, USA). The Gene Ruler DNA ladder mix (Fermentas, Lithuania) was used as molecular weight marker.
PCR products were separated in 0.8% (w/v) agarose gels in 1XTAE buffer, and visualized after staining with ethidium bromide using a GelDoc 2000 system (Bio-Rad, Hercules, USA). The Gene Ruler DNA ladder mix (Fermentas, Lithuania) was used as molecular weight marker.
8
Genotyping Arabidopsis thaliana Accessions
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Col-0 sequences are corresponding to the TAIR10 version of the reference genome. To genotype plants, we used two molecular markers, one located within the 5'-UTR of TAD3-1 (MSAT5.08448), amplifying 142 bp in Col-0 and 138 bp in Nok-1, and another one that co-segregates with the interval of 14–15 Mb at chromosome 1 (MSAT1.15597; S1 Fig ), and amplifying 128 bp in Col-0 and less in Nok1 (≈120 bp). The GeneRuler DNA Ladder Mix (Ref SM0331, Thermo) is the DNA ladder used for all figures.
9
Origami Reaction Self-Assembly Protocol
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The thermal condition for self-assembly in origami reaction was set using C1000 thermal cycler (Bio-Rad, California, USA). Transmission electron microscopy was done using Philips EM028 TEM, Aachen, Germany. The micrographs were obtained by JPK-AFM (JPK Instruments AG, Berlin, Germany). Mica was prepared from Nanotechnology Systems Corporation, Tehran, Iran. M13 mp18 phage genome and T4 DNA ligase were purchased from New England Biolabs (Massachusetts, USA). Desired single-stranded oligonucleotides were synthesized and desalted by Sigma–Aldrich Chemie GmbH (Munich, Germany). Quantum Prep Freeze ‘N Squeeze DNA gel-extraction spin columns were from Bio-Rad. SYBR Gold nucleic-acid gel stain was purchased from Molecular Probes Inc. (Eugene, Oregon, USA). GeneRuler DNA Ladder Mix was from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).
10
Quantifying DNA from Decellularized Heart
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Membranes were air dried and weighed and total DNA from both native and decellularized HCM (dHCM) was extracted using the DNeasy Blood and Tissue Kit (#69504, Qiagen, Hilden, Germany), according to manufacturer’s instructions. The quantification of double-stranded DNA (dsDNA) was performed using the Quant-iT PicoGreen dsDNA Assay Kit (#P7589, Invitrogen, Carlsbad, CA, USA), according to manufacturer’s instructions. Electrophoresis was performed to assess the size of the DNA fragments, using a 1% agarose gel and GeneRuler DNA Ladder Mix (#SM0334, Thermo Fisher Scientific, Waltham, MA, USA). Four independent samples were used in each condition.
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