To validate that DNA samples were of the correct and single strain of origin, samples were PCR amplified for DP496, a microsatellite locus previously identified in Colbourne et al. (2004) and demonstrated to have discriminating allelic patterns between these two D. pulicaria strains (Wilson and Hay 2007 ; Chislock et al. 2019a ). Primer sequences were obtained from the Daphnia Genomics Consortium, wfleabase (http://wfleabase.org/genomics/microsatellite/ ) (Colbourne et al. 2004 , 2005 (link)). PCR reactions were carried out in 10 µl volumes using 5 µl of 2X GoTaq Green PCR Master Mix [Promega, USA], 0.3 µl of 10 µM forward and reverse primers (0.3 µM final concentration), 3.65 µl of water, and 0.75 µl of DNA (21 ng). The thermocycler program for the PCR began with a 2 min denaturation cycle at 95°C, followed by 35 cycles of 20 s at 95°C, 20 s at 50°C, and 20 s at 72°C, and a final extension cycle for 10 min at 72°C. DNA from a single individual of each strain was used as positive controls, and water instead of DNA was used as a no template control. Five microliters of PCR products were visualized on a 3% agarose gel made with 0.5X TAE and 1 µl of GelGreen Nucleic Acid Stain [Biotium, USA] to confirm that the allelic patterns for the genome samples were consistent with the positive controls for the target strains.
Gelgreen nucleic acid stain
Manufactured by Biotium
Sourced in United States, United Kingdom
GelGreen Nucleic Acid Stain is a fluorescent dye used for the detection and visualization of nucleic acids, such as DNA and RNA, in agarose gels. It is a sensitive and environmentally friendly alternative to traditional nucleic acid stains like ethidium bromide.
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Lab products found in correlation
Bigdye terminator 3.1 kit, by Thermo Fisher Scientific (2 mentions)
Dreamtaq green dna polymerase, by Thermo Fisher Scientific (2 mentions)
Genie explorer software, by OptiGene (2 mentions)
Genie 3, by OptiGene (2 mentions)
Sybr green 1 nucleic acid gel stain, by Thermo Fisher Scientific (2 mentions)
Gotaq green pcr master mix, by Promega (1 mentions)
Verapamil, by Merck Group (1 mentions)
Nelfinavir, by Merck Group (1 mentions)
Cytochalasin b, by Merck Group (1 mentions)
Fluorescein diacetate fda, by Thermo Fisher Scientific (1 mentions)
Ketoconazole, by Merck Group (1 mentions)
Vincristine, by Merck Group (1 mentions)
Quinidine, by Merck Group (1 mentions)
Bisbenzimide h 33258, by Merck Group (1 mentions)
Sodium fluorescein, by Merck Group (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Benzbromarone, by Merck Group (1 mentions)
Riddelliine, by Phytolab (1 mentions)
Lasiocarpine, by Phytolab (1 mentions)
Cyclophosphamide, by Thermo Fisher Scientific (1 mentions)
13 protocols using gelgreen nucleic acid stain
1
Validating Daphnia pulicaria Strain Identity
2
Evaluation of Cell Cytotoxicity Assays
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PAs (europine (CAS# 570-19-4; assay 100%), riddelliine (CAS# 23,246-96-0; assay ≥ 98%) and lasiocarpine (CAS# 303-34-4; assay 100%) were obtained from PhytoLab (Vestenbergsgreuth, Bayern, Germany); Cyclophosphamide (CAS# 6055–19-2; assay ≥ 97%) was from Alfa Aesar (Karlsruhe, Germany). Gel Green Nucleic Acid stain was obtained from Biotium (Darmstadt, Germany). Fluorescein diacetate (FDA; CAS# 596-09-8) was from Invitrogen (Germany). Dimethylsulfoximide (DMSO; ≥ 99.8%), bisBenzimide H33258 (CAS# 23,491–45-4; assay ≥ 98%), diazabicyclo-octane (DABCO), ketoconazole (CAS# 65,277-42-1; assay ≥ 98%), quinidine (CAS# 56-54-2; assay ≥ 97%), verapamil (CAS# 152-11-4; assay ≥ 99%), nelfinavir (CAS# 159,989-65-8; assay ≥ 98%), benzbromarone (CAS# 3562-84-3; assay ≥ 95%), vincristine (CAS# 2068-78-2; assay ≥ 97%), cytochalasin B (CAS# 14,930-96-2), ethidium bromide (CAS# 1239-45-8; assay ≥ 95%) and sodium fluorescein (CAS# 518-47-8; assay ≥ 95%) were from Sigma-Aldrich (Steinheim, Germany). Calcein-acetoxymethyl ester (Calcein-AM; CAS# 148,504-34-1; assay ≥ 95%) was from Cayman Chemical Company (Germany). Cell culture media and reagents were all from Sigma-Aldrich (Steinheim, Germany), except foetal bovine serum, which was from Biochrom (Berlin, Germany).
3
Neutral Comet Assay for DNA Damage
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DNA double strand breaks were detected using neutral CometAssay Kit (Trivagen, MD, USA) following the manufacturer’s instructions. Briefly, control and Mortaparib-treated cells were harvested and mixed with pre-warmed (37 °C) agarose, and layered immediately onto Cometslide™ (Trivagen). Slides were placed in dark at 4 °C for 1 h and immersed in lysis solution at 4 °C overnight. Excess of lysis solution was removed and slides were subjected to electrophoresis using Neutral Electrophoresis Buffer™ (Trivagen). The slides were then washed with 70% ethanol at room for 30 min temperature followed by drying at 37 °C for 10–15 min. GelGreen® nucleic acid stain (Biotium, CA, USA) was added onto slides and washed thrice with PBS. Comet formation was observed and recorded under Zeiss Axiovert 200 M microscope (Carl Zeiss Microimaging, Thornwood, NY).
4
Plasmid DNA Conformational Analysis
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The pDNA was incubated for 3 h at RT with the following solvent mixtures: DCM, DCM + 0.01% sodium cholate, DCM + 0.3% sodium cholate, DCM + 0.6% sodium cholate, DCM + 1% sodium cholate, DCM + 1% PVA, DCM + 1% (41% Tween 80 + 59% Tween 20) and DCM + 1% Triton X-100, mimicking the conditions during the double emulsion procedure. Subsequent loading onto a 1% agarose gel in TBE buffer (8.9 mM Tris, 8.9 mM boric acid, 0.2 mM EDTA, pH 8.4) containing Gel Green nucleic acid stain (Biotium, Fremont, CA, USA) and electrophoresis resolved the different conformations present in each sample. Purified pDNA stock in DNAse-free Milli-Q water was used as the control, confirming the exclusive presence of the supercoiled functional conformation in the sample. Adequate separation of the bands for supercoiled, linear, and open circular plasmid DNA was obtained by running the electrophoresis gel for at least 40 min at 60 V. Images were acquired using a UV transilluminator (Vilber Lourmat, TCX-20C, Collégien, France).
5
Fungal Strain Identification via ITS Sequencing
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Growing mycelia of individual fungal strains were maintained in pure cultures for 7 days on PDA medium for genomic DNA extraction. Genomic DNA was extracted using a modified CTAB (hexadecyltrimethylammonium bromide) method, described earlier [27 ]. The DNA extracts were stored at – 20 °C until used. Species identification was done on the basis of the sequences of the Internal Transcribed Spacers of the ribosomal DNA region (ITS1-ITS2).
Polymerase chain reactions were performed as described previously by Kozłowska et al. [9 ], using DreamTaq Green DNA polymerase (Thermo Scientific, Espoo, Finland). The PCR amplification was done using ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) primers. Amplicons were electrophoresed in 1.5% agarose gel (Invitrogen) with GelGreen Nucleic Acid Stain (Biotium, Inc.).
For sequence analysis, PCR-amplified DNA fragments were purified as described previosly by Kozłowska et al. [9 ], DNA fragments were labelled using a forward primer and the BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to the producer’s recommendations, and precipitated with 96% ethanol. Sequence reading was performed using Applied Biosystems equipment. Sequences were analysed using the BLASTn algorithm.
Polymerase chain reactions were performed as described previously by Kozłowska et al. [9 ], using DreamTaq Green DNA polymerase (Thermo Scientific, Espoo, Finland). The PCR amplification was done using ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) primers. Amplicons were electrophoresed in 1.5% agarose gel (Invitrogen) with GelGreen Nucleic Acid Stain (Biotium, Inc.).
For sequence analysis, PCR-amplified DNA fragments were purified as described previosly by Kozłowska et al. [9 ], DNA fragments were labelled using a forward primer and the BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to the producer’s recommendations, and precipitated with 96% ethanol. Sequence reading was performed using Applied Biosystems equipment. Sequences were analysed using the BLASTn algorithm.
6
Fungal DNA Extraction and Identification
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A modified method using CTAB (hexadecyltrimethylammonium bromide) was applied for genomic DNA extraction, as described earlier58 (link). Species identification was performed on the basis of the sequence analysis of the Internal Transcribed Spacers of the ribosomal DNA region (ITS1-ITS2).
Polymerase chain reactions (PCRs) were performed as described earlier27 (link) using DreamTaq Green DNA polymerase (Thermo Scientific, Espoo, Finland). For the PCR amplification specific primers were used: ITS4 – forward primer (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 – reverse primer (5′-GGAAGTAAAAGTCGTAACAAGG-3′)59 . Amplicons were separated in 1.5% agarose gel (Invitrogen) with GelGreen Nucleic Acid Stain (Biotium, Inc.).
For sequence analysis, PCR-amplified DNA fragments were purified as described earlier60 (link). DNA fragments were labelled using a forward primer and the BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to the producer’s recommendations and precipitated with 96% ethanol. Sequence reading was performed using Applied Biosystems equipment. Sequences were analysed using the BLASTn algorithm against the GenBank database-deposited reference sequences.
Polymerase chain reactions (PCRs) were performed as described earlier27 (link) using DreamTaq Green DNA polymerase (Thermo Scientific, Espoo, Finland). For the PCR amplification specific primers were used: ITS4 – forward primer (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 – reverse primer (5′-GGAAGTAAAAGTCGTAACAAGG-3′)59 . Amplicons were separated in 1.5% agarose gel (Invitrogen) with GelGreen Nucleic Acid Stain (Biotium, Inc.).
For sequence analysis, PCR-amplified DNA fragments were purified as described earlier60 (link). DNA fragments were labelled using a forward primer and the BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to the producer’s recommendations and precipitated with 96% ethanol. Sequence reading was performed using Applied Biosystems equipment. Sequences were analysed using the BLASTn algorithm against the GenBank database-deposited reference sequences.
7
Quantitative RT-PCR Analysis of Gene Expression
8
Agarose Gel Electrophoresis Protocol
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First, 1% gel agarose (Hydragene, #R9012LE) was dissolved in TAEX1 (dissolved with DDW from TAEX50 (Biolab, #2050237500)) and then carefully heated in a microwave. When the solution was completely clear, 8 μL of GelGreen nucleic acid stain (Biotium, #41005) was added. The solution was poured into a mold and the gel comb was immediately inserted. The gel was cooled to room temperature for 20 min and then gently placed in the TBEX1 buffer-filled box. All samples were loaded in their wells including DNA markers, and an electrical current was applied via the power supply to the rear. After about 30 min, the gel was removed for imaging under ultraviolet light.
9
LAMP Amplification Detection Methods
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The LAMP amplification results were detected with three different methods in a laboratory setting: 1. on 1% TBE agarose gel stained with GelGreen® Nucleic Acid Stain (Biotium, Fremont, CA) for visual observation; 2. with SYBR™ Green 1 nucleic acid gel stain (Invitrogen, Carlsbad, CA) for observation under UV light; and 3. using Genie® III (OptiGene, Horsham, WS, UK) instrument by obtaining the amplification curves and analyzing data using Genie Explorer software (OptiGene, Horsham, WS, UK). All reactions were repeated at least three times.
10
In Vitro Cre Recombination Assay
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Two direct loxP repeats or other variants of loxP/M7 sites separated by a ∼0.5 kb spacer were cloned between the XbaI and SphI sites of the pBAD33 plasmid. The 0.7 kb DNA substrate for in vitro recombination assays was generated by polymerase chain reaction (PCR) amplification with pBAD-forward and pBAD-reverse primers. One microgram of the DNA substrate was incubated with 1 μM Cre in 10 μl of 50 mM Tris–HCl, pH 7.8, 50 mM NaCl and 10 mM MgCl2 for 12 h at 37°C. The total amount of protein used is the same across all in vitro assays. Reactions were stopped by incubation at 98°C for 20 min. Reactions were analyzed on 2% agarose gels and visualized by staining with GelGreen nucleic acid stain (Biotium).
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