A total of 100 ng of 5.7 kilobase pair expression vector, pET-15b (Novagen) were mixed with appropriate amounts of polymer to achieve the desired N/P ratio, and diluted in TE buffer (20 mM Tris-acetate/0.5 mM EDTA pH 8.2) to obtain a final volume of 10 µL. A volume of 2 µL of Blue 6X loading dye (Fisher Scientific) was added, after which 12 µL was run on a 0.7% wt/vol agarose gel (50 V) in the same TE buffer. DNA was visualized with SYBER Safe (Life Technologies).
Syber safe
Manufactured by Thermo Fisher Scientific
Sourced in United States
Syber Safe is a nucleic acid stain used for the detection and quantification of DNA and RNA in electrophoresis gels. It is a sensitive and specific fluorescent dye that binds to nucleic acids, allowing for visualization under ultraviolet (UV) or blue light illumination.
Automatically generated - may contain errors
Lab products found in correlation
Molecular imager chemidoc xrs system, by Bio-Rad (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Blue 6x loading dye, by Thermo Fisher Scientific (1 mentions)
Random hexamer, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by New England Biolabs (1 mentions)
Emeraldamp gt pcr master mix, by Takara Bio (1 mentions)
High glucose dmem f12, by Thermo Fisher Scientific (1 mentions)
Image station 4000mm pro, by Carestream (1 mentions)
Veriti 96 well thermocycler, by Thermo Fisher Scientific (1 mentions)
Thermal cycler, by Eppendorf (1 mentions)
Gel purification, by Qiagen (1 mentions)
Chemdoc, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Nuclease free water, by Merck Group (1 mentions)
Le agarose, by Lonza (1 mentions)
Dynazyme reaction buffer, by Thermo Fisher Scientific (1 mentions)
Dynazyme 2 polymerase, by Thermo Fisher Scientific (1 mentions)
Mastercycler gradient temperature cycler, by Eppendorf (1 mentions)
1 kb dna ladder, by Thermo Fisher Scientific (1 mentions)
27 protocols using syber safe
1
Agarose Gel Electrophoresis of Plasmid DNA
2
Isolation and Analysis of Meiotic mRNA
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Two hundred nanograms of polyA-selected mRNA from 5 h meiotic cells were denatured at 70 °C for 2 min and placed on ice. Next, 4 μl MazF buffer and 0.5 μl RNase inhibitor (NEB, M0314L) were added, and the volume was made up to 19 μl with RNase-free water. Next, 1 μl (20 U) of MazF (TaKaRa 2415A) were added and the samples were incubated at 37 °C for 2 h before ethanol precipitation with glycogen blue as described above. Next, RNA was resuspended in 8 μl RNAse-free water and cDNA was synthesized using SuperScript III with random hexamers (Life Technologies 18080–051) according to the manufacturer’s instructions. Two microliters of the resulting cDNA were used in subsequent PCR reactions using EmeraldAmp GT PCR master mix (TaKaRa RR310), and the products were run on a 2% agarose TBE gel stained with SYBER safe (Life Technologies S33102).
3
Nile Tilapia Spermatogonial Stem Cell Isolation
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Nile tilapia SSCs were obtained through primary culture from adult fish testis12 (link)22 23 (link)24 , and 106 cells per dish were exposed to 1 mL of lentiviral solution (titre = 6.5 × 105 TU/mL). The cells were incubated at 33 °C and 5% CO2 for 24 h, and the aliquot was submitted to flow cytometry in order to assess the percentage of fluorescent cells before selection. Blasticidin (6 μg/mL) was added to the supplemented DMEM/F12 culture medium (High Glucose DMEM/F12 (THERMO), 10% foetal bovine serum, 0.1 mM non-essential amino acids, 6 mM L-glutamine, 1 mM sodium pyruvate, and 1% penicillin-streptomycin) and the cells were maintained for one month12 (link). After this time, their fluorescence was assessed by flow cytometry. Cell genomic DNA was extracted and submitted to PCR in order to confirm the presence of the DsRed2 encoding sequence on a 1% agarose gel with Syber Safe (LIFE TECHNOLOGIES). Twenty larvae (2–3 days after hatching) were anesthetised and microinjected in the coelomic cavity with approximately 2 μL of cell suspension containing 5 × 104 SSCs/μL, using a micromanipulator (NARISHIGE) and glass needles prepared with GD-1 capillaries (NARISHIGE) as previously described25 (link). The red fluorescence was monitored through fluorescence and confocal microscopy, and the sperm samples from male recipients were analysed by flow cytometry and fluorescence microscopy.
4
Molecular Screening of Rice Blast Resistance
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The PCR was performed using a total volume of 10 µl reaction mixture, containing 3 µl of 30 ng of template DNA and 0.5 µl of 5 Pico moles of each primer (Invitrogen), 1 µl of reaction buffer, 1 µl of dNTPs and 0.2 µl of Taq DNA polymerase. The PCR profile used was one cycle of initial denaturation at 95 °C for 5 min, followed by 35 cycles of amplification (each cycle of 1 min denaturation at 95 °C, 30 secs annealing at 55 °C, 30 secs extension at 72 °C), followed by a final extension of 72 °C for 7 min and stored at 4 °C. The amplified products were resolved in 3.5% Agarose gel with 0.5 X TBE buffer containing 0.1 μg/ml of Syber safe (S33102, Invitrogen) and documented in the Gel documentation system21 (link). Gel scoring was carried out based on the donor and recurrent parent banding patterns and their corresponding product sizes. The allele corresponding with Matatag 1 was considered resistant allele. Similarly, allele matched with recipient parent BRRI dhan71 scored as a susceptible allele. Whereas if both the alleles (susceptible and resistant) were observed in one sample, they were considered heterozygotes.
5
Analyzing Protein-DNA Interactions of Tudor-SN
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A 447 bp genomic region containing the Tudor-SN locus was subcloned into pTST101 to make pTST101-447pos (oligonucleotides are shown in Supplementary Table S4 ). pTST101-447pos was digested by NdeI, HindIII, and SalI resulting in three linear fragments, including DNAtudor (1627 bp long dsDNA fragment containing the 447 bp Tudor-SN locus) and two additional dsDNA fragments (750 and 4025 bp). Restricted pTST101 (1.7 nM) was incubated with increasing amounts of purified BEAF32, CP190 or Chromator in 150 mM NaCl, 30 mM Tris/HCl pH 7.4, 5 mM mercaptoethanol. A gel loading buffer (50% glycerol, 50 mM Tris/HCl pH 7.4) was added and the DNA-protein mixture was directly analyzed in a 1% TAE agarose gel. DNA was labeled using Sybersafe (Invitrogen) and visualized on a gel imaging system (Image Station 4000 MM Pro–Carestream Molecular Imaging). No difference in binding specificity was observed when DNA competitors (e.g. dIdC) were added to the protein-DNA mix. For super-shift assays, the 447 bp Tudor-SN locus (chromosome 3L: 264375–264822) was PCR amplified from S2 Drosophila genomic DNA. Purified proteins were added to the DNA in a reaction mixture in a total volume of 20 µl and incubated for 10 min on ice. A gel loading solution (50% glycerol, 50 mM Tris/HCl pH 7.4) was added and the DNA-protein mixture was directly analyzed on a 2% TAE agarose gel.
6
IS900 PCR-based Mycobacterium avium Identification
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IS900 PCR [36 (link)] was used for identification of all Map isolates from the AJDRC and used in this study (additional File 1 : Table S1). The PCRs were performed using 1 μl of extracted DNA in a PCR mix containing 1 μM of primers IS900/150C and IS900/921, 10 mM Tris hydrochloride, (pH 8.3), 50 mM potassium chloride, 1.5 mM magnesium chloride, 0.01% gelatin, 200 μM of each of the nucleotides dATP, dTTP, dGTP and dCTP, and 2.5 U of Taq polymerase. The assay was performed on a Veriti 96 well thermo cycler (Thermo Fischer Scientific, Massachusetts, USA) under the following conditions: 35 cycles of denaturation at 94 °C for 10 s, annealing at 60 °C for 10 s, and extension at 72 °C for 10 s. The expected 229 bp IS900 PCR amplicon was visualised on a 2% agarose gel stained with Syber Safe (Invitrogen).
7
PCR Verification of M13 Colonies
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Polymerase chain reaction (PCR) was performed to confirm that the blue colonies isolated from faeces were M13. The reactions were conducted for a final volume of 10 μL, containing 1 μL of the DNA sample, 10 pmol/μL of each primer (forward: 5ʹ-GTAAAACGACGGCCAG-3ʹ, reverse: 5ʹ-CAGGAAACAGCTATGAC-3ʹ) (Invitrogen), 0.25 μl of 20 mM of the mix of dNTPs (Invitrogen), 0.25 μL of Taq (5U/μL) (Invitrogen). The samples were submitted to the following amplification cycles: initial denaturation at 95 °C for 5 min, 35 cycles at 94 °C for 40 s, 56 °C for 40 s, 72 °C for 50 s, and a final extension at 72 °C for 10 min. All PCR reactions were performed on a thermal cycler (Eppendorf), and the amplified products were separated via 1.5% agarose gel electrophoresis. The gel was stained with Syber Safe (Invitrogen) and visualised in UV translucent vessels.
8
Polyacrylamide Gel Electrophoresis of PCR Products
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Products of PCR amplifications, with or without gel purification (Qiagen) were run on denaturing urea (8 M) polyacrylamide gel (8% w/v) in 1X TBE buffer following heat denaturation of samples at 95°C for 5 min and subsequent snap-cooling on ice. The electrophoresis was carried out at 25 W after pre-running the gel for 1 h. Typically, the gel was stained in 1X TBE containing 0.02% (v/v) SyberSafe (Invitrogen) and then imaged using ChemDoc (Bio-rad). Band intensity was analyzed using Image Lab software (Bio-Rad). When required, bands were eluted with a solution of 0.3M NaCl and ethanol precipitated.
9
Amplification of Environmental Gene Fragments
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pmoA, mxaF, dsrB and narG gene fragments were amplified from the three parallel samples of DNA and cDNA obtained from untreated fracture fluid as well as from the treated samples. PCR was performed in 50 μL reaction volumes containing 4 μL template, 1 × Dynazyme Reaction Buffer (Finnzymes, Vantaa, Finland), 62.5 μM dNTP (final concentration) (Finnzymes, Vantaa, Finland), 0.2 μM (final concentration) of each forward and reverse primer (Eurogentec, Belgium) (Table 1 ), 1 U Dynazyme II polymerase (Finnzymes, Vantaa, Finland) and nuclease free water (Sigma, St. Louis, MO, USA). All PCR reactions were carried out on a Mastercycler gradient temperature cycler (Eppendorf, Hamburg, Germany) using the following conditions, 95 °C initial denaturation for 5 min followed by 40 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, with final extension step at 72 °C for 10 min, with the exception of an annealing temperature of 58 °C being used for narG. PCR products were visualized with agarose gel electrophoresis on a 1% agarose gel (LE-agarose, Lonza, Basel, Switzerland) stained with 1 × SyberSafe (Invitrogen, Carlsbad, CA, USA) in 1 × SB-buffer [18 (link)] at 300 V for 15 min.
10
Identification of Invasion-Related Genes in P. aeruginosa
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The detection of invasion-related genes belonging to the T1SS, T2SS and T3SS was performed via PCR in a Veriti thermal cycler (Applied Biosystem, Foster City, CA, USA) according to the protocol described by Rodrigues et al. [32 (link)]. Visualization of PCR products was performed via 1.5% agarose gel electrophoresis at 110 V for 45 min in TAE 1× buffer (89 nM Tris-borate and 2 mM EDTA pH 8.0). As molecular weight marker, 1 Kb DNA ladder (Invitrogen™) was used, gel stained with SyberSafe (Invitrogen™, Carlsbad, CA, USA)) and differentiation of bands visualized under ultraviolet light. In addition, the pigment production and mucoid phenotype of P. aeruginosa isolates were verified by observing bacterial growth on MHA agar plates and slants.
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