The PCR reactions were performed to detect the DNA of bacteriophages vB_SenM-2 and vB_Sen-TO17 in the plasma of chickens. For this purpose, 10 µl of Color Taq PCR Master Mix (2×), 2 µl of specific forward and reverse primers each (final concentration of 1 µM), 5 µl of nuclease-free water, and 1 µl of matrix DNA (concentration 10 ng/µl) were mixed. The reaction was conducted in Mastercycler® nexus (Eppendorf, Germany) with the following parameters: number of cycles—30; denaturation—15 s, 94 °C; annealing—15 s, 55 °C; extension—60 s, 72 °C. Then, the samples were loaded into wells of 1.5% agarose gel (agarose solution in Tris-octane-EDTA buffer (Bioshop, Canada) supplemented with SimplySafeTM (EURx, #E4600-02). Electrophoresis was set at 100 V for 30 min. DNA was visualized by UV light using a gel documentation system (FastGene FAS-DIGI PRO, Nippon Genetics Europe, Germany) with the following parameters: aperture 5.6 AV, exposure 1/40 TV, ISO 800.
Simplysafe
Manufactured by EURx
Sourced in Poland, United States
SimplySafe is a compact and portable laboratory equipment designed for basic liquid handling tasks. It provides a simple and efficient solution for tasks such as pipetting, mixing, and dispensing small volumes of liquids in a laboratory setting. The device features a straightforward user interface and is intended for general laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
G box, by Syngene (6 mentions)
Hd polymerase, by Takara Bio (4 mentions)
Pcr buffer, by Takara Bio (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Rnase free dnase set, by Qiagen (3 mentions)
Fastgene fas digi pro, by Nippon Genetics (2 mentions)
Mastercycler nexus, by Eppendorf (2 mentions)
Dreamtaq dna polymerase, by Thermo Fisher Scientific (2 mentions)
T100 thermal cycler, by Bio-Rad (2 mentions)
Chemidoc, by Bio-Rad (2 mentions)
Gel out concentrator, by A&A Biotechnology (2 mentions)
Tri reagent, by Merck Group (2 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (2 mentions)
Dreamtaq polymerase, by Thermo Fisher Scientific (2 mentions)
Universal rna purification kit, by EURx (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nuclease free water, by EURx (1 mentions)
Taq dna polymerase, by Promega (1 mentions)
Mag bind plant dna 96 kit, by Omega Bio-Tek (1 mentions)
Multidoc it imaging system, by Analytik Jena (1 mentions)
50 protocols using simplysafe
1
Detecting Bacteriophage DNA in Chicken Plasma
2
Bacterial Genomic DNA Extraction and PCR Amplification
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Total genomic DNA from L. bulgaricus strain 43 was extracted with the GeneMATRIX Bacteria & Yeast Genomic DNA Purification Kit (EURx® , Gdansk, Poland).
PCR amplification of the fragment (containing the β-gal gene) was performed in QB-96 Satellite Gradient Thermal Cycler (LKB Vertriebs GmbH, Vienna, Austria).
PCR reactions consisted of a 150 ng DNA template, 0.4 µM of primers, 12.5 μL of Premix Ex Taq Hot Start Version 2.0 (Clontech Laboratories, Inc., A Takara Bio Company (Mountain View, CA, USA), and nuclease-free water (EURx® , Gdansk, Poland) to a 25 µL final volume. The next temperature profile was as follows: initial denaturation, 3 min at 95 °C; 35 cycles: 10 s denaturation at 98 °C, 30 s annealing at 60 °C, and 1 min elongation at 72 °C; final elongation—5 min at 72 °C. A gradient of annealing temperatures from 54.2 °C to 62.7 °C showed no loss of quality in the PCR product. Two pairs of primers, listed in Table 2 , were used for sequencing the complete PCR fragment (Macrogen Inc., Amsterdam, The Netherlands).
DNA fragments were visualized using gel electrophoresis on agarose (AlfaAesar, Kandel, Germany), in TAE buffer (40 mM of Tris-base, 20 mM of acetic acid, 1 mM of EDTA), and stained with SimplySafeTM (EURx, Gdansk, Poland).
PCR amplification of the fragment (containing the β-gal gene) was performed in QB-96 Satellite Gradient Thermal Cycler (LKB Vertriebs GmbH, Vienna, Austria).
PCR reactions consisted of a 150 ng DNA template, 0.4 µM of primers, 12.5 μL of Premix Ex Taq Hot Start Version 2.0 (Clontech Laboratories, Inc., A Takara Bio Company (Mountain View, CA, USA), and nuclease-free water (EURx
DNA fragments were visualized using gel electrophoresis on agarose (AlfaAesar, Kandel, Germany), in TAE buffer (40 mM of Tris-base, 20 mM of acetic acid, 1 mM of EDTA), and stained with SimplySafeTM (EURx, Gdansk, Poland).
3
Amplification and Identification of 16S rDNA
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The amplification reaction of the longer 16S rDNA fragment employing the primers 16Sar and 16Sbr described by Palumbi (1996) was performed in a total volume of 20 µl, with Promega (Madison, WI), Buffer 1x, 2.5 mM MgCl2, 0.25 mM dNTPs, 20 pmol of each primer, approximately 20 ng of template DNA and 1 U of DNA Taq polymerase (Promega), and the following PCR conditions: initial denaturing at 95ºC for 5 min, 35
cycles of denaturing at 94ºC for 1 min, annealing at 55ºC for 1 minute, extension at 72ºC for 2 minutes and final extension at 72ºC for 7 minutes.
For the new species-specific primers the PCR conditions were the same except for the annealing temperature that was 50ºC.
PCR products were visualized under UV light on a 1% agarose gel stained with SimplySafe TM from EURx.
Sequencing was performed by the DNA sequencing service Macrogen Europe.
The sequences obtained were compared with international databases employing the program BLASTn within the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) for confirming species identification.
cycles of denaturing at 94ºC for 1 min, annealing at 55ºC for 1 minute, extension at 72ºC for 2 minutes and final extension at 72ºC for 7 minutes.
For the new species-specific primers the PCR conditions were the same except for the annealing temperature that was 50ºC.
PCR products were visualized under UV light on a 1% agarose gel stained with SimplySafe TM from EURx.
Sequencing was performed by the DNA sequencing service Macrogen Europe.
The sequences obtained were compared with international databases employing the program BLASTn within the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) for confirming species identification.
4
PCR-RFLP Analysis of Genetic Polymorphism
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The polymorphism was assessed by PCR-restriction fragment length polymorphism (RFLP). Each PCR mix (25µl) contained 150ng genomic DNA, and PCR buffer (Clontech Laboratories, USA), dNTPs mix (0,25mM), HD polymerase (Clontech Laboratories, USA) and primers (10µM of each). The mix was heated to 94 o C for 5min and underwent 35 cycles of ampli cation: denaturation 98 0 C for 10s, annealing The PCR products were digested with MwoI (HpyF10VI) restriction enzyme (Thermo Fisher Scienti c, USA) for 16 hours at 37 o C. RFLP products were analyzed on 3% agarose gel and stained with SimplySafe (Eurx, Poland) and visualized in G:Box (Syngene, Great Britain). T or C alleles were identi ed by the presence of 203bp (TT genotype) or 158bp (CC genotype) fragments, respectively. Heterozygous TC genotype showed the presence of two bands -158bp and 203bp (Fig. 3a). An independent PCR analysis was carried out for each sample.
5
PSMA6 Polymorphism Analysis by PCR-RFLP
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For analysis of PSMA6 polymorphism PCR-RFLP method was applied according to validated protocol of Bachmann et al. 2010 28 . PSMA6 gene fragment length of 100 bp was ampli ed in PCR reaction using following primers:
-forward 5'-CTG GTG CGG GAG CTA CGG G-3' -reverse 5'-AAT GGT AAT GTG GCG GTC AAA AC-3' Each PCR mixture (25µl) contained 100ng genomic DNA and PCR buffer (Clontech), dNTPs mixture (0.25mM), HD polymerase (Clontech) and primers (10µM of each). The touchdown PCR method was used. The mixture was heated 95 o C for 5min and underwent 14 cycles of ampli cation: denaturation The PCR product was digested with RsaI (Thermo Fisher Scienti c) for 16 hours at 37 0 C producing two fragments of 50 bp or one fragment 100 bp for presence of G or C allele, respectively. RFLP products were analyzed on 3% agarose gel and stained with SimplySafe (Eurx, Poland) and visualized in G:Box (Syngene, Great Britain) (Fig. 3b). An independent PCR analysis was carried out for each sample.
-forward 5'-CTG GTG CGG GAG CTA CGG G-3' -reverse 5'-AAT GGT AAT GTG GCG GTC AAA AC-3' Each PCR mixture (25µl) contained 100ng genomic DNA and PCR buffer (Clontech), dNTPs mixture (0.25mM), HD polymerase (Clontech) and primers (10µM of each). The touchdown PCR method was used. The mixture was heated 95 o C for 5min and underwent 14 cycles of ampli cation: denaturation The PCR product was digested with RsaI (Thermo Fisher Scienti c) for 16 hours at 37 0 C producing two fragments of 50 bp or one fragment 100 bp for presence of G or C allele, respectively. RFLP products were analyzed on 3% agarose gel and stained with SimplySafe (Eurx, Poland) and visualized in G:Box (Syngene, Great Britain) (Fig. 3b). An independent PCR analysis was carried out for each sample.
6
Genotyping Mapping Population via DArTseq
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Genomic DNA for SNP-DArT (DArTseq) and silico-DArT genotyping was isolated from 329 plants of the G38A mapping population and the two parental lines using the Mag-Bind Plant DNA 96 Kit (Omega Bio-Tek, USA) in accordance with the manufacturer’s instructions. One hundred milligrams of ground leaf tissue was used for each analyzed plant. The isolated DNA was dissolved in 100 μl elution buffer. The DNA yield and purity were estimated using a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, USA) and electrophoresed in 1% agarose gel stained with SimplySafe (Eurx, Poland). The DNA concentration was adjusted to 100 ng and then samples were sent to the Diversity Arrays Technology Pty Ltd. (Bruce, Australia), where genotyping was performed employing the DArTseq 1.0 technology developed by Cruz et al. [29 (link)] (Table S2 and S3 ).
Marker annotation was performed by mapping the sequences and chromosome positions to the Lo7 rye genome [15 (link)]. All gene names mentioned in the text are taken from Rabanus-Wallace et al. [15 (link)].
Marker annotation was performed by mapping the sequences and chromosome positions to the Lo7 rye genome [15 (link)]. All gene names mentioned in the text are taken from Rabanus-Wallace et al. [15 (link)].
7
ISSR Markers For Genetic Diversity
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Six primers of ISSR markers were selected (Gupta et al. 1994; (link)Stepansky et al. 1999 ) (Table 1). The analysis with ISSR markers was based on protocols developed by Żabicka et al. (2020) ; the same reagents and laboratory equipment were used. The PCR products were separated in 1% agarose gel with 1X TBE and SimplySafe (EURx Sp. z o.o. Gdansk, Poland) for about 90 min at 120 V. Band patterns of 38 samples were captured with a MultiDoc-It™ Imaging System with VisionWorks® LS Analysis Software (UVP, Upland, CA, USA). PCR reaction cycle were repeated on selected samples to test reproducibility of obtained results.
Split phylogenetic network (NeighborNet) was constructed in SplitsTree v. 4.6 (Huson and Bryant 2006) (link) based on Dice coefficient to evaluate the relationships between examined individuals, bootstrap was calculated on 1000 replicates.
Split phylogenetic network (NeighborNet) was constructed in SplitsTree v. 4.6 (Huson and Bryant 2006) (link) based on Dice coefficient to evaluate the relationships between examined individuals, bootstrap was calculated on 1000 replicates.
8
Molecular Identification of Enteroviruses and Adenoviruses
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All of the amplified PCR products were analyzed by electrophoresis in 2% agarose gel stained with SimplySafe (EURx, Poland) and visualized under UV light. PCR products were then extracted and purified from the gel using QIAquick gel extraction kit (Qiagen Ltd, Crawley, UK). The second round PCR products of Enteroviruses and Adenoviruses were sequenced using the ABI 3110 DNA SEQUENCER (Applied Biosystems, Foster City, CA). The partial nucleotide sequences of VP1 and hexon regions were aligned by the CLUSTALW and phylogenetic tree analysis was obtained by the neighbor-joining method under the Kimura-two-parameter distance model by the software package of MEGA7 (www.megasoftware.net ). The reference strains used for comparisons with sequences from this study were obtained from GenBank.
In order to identify Enterovirus serotypes, pairwise comparison of the VP1 amplicon sequence was performed with the complete database of VP1 sequences of all known EV serotypes using the BLAST program (www.ncbi.nlm.gov/BLAST ) from the Gen-Bank. At least 75% identity in the VP1 sequence is required to put strains in the same serotype (18 (link)).
Types of Adenoviruses were determined by comparing the sequences obtained from the hexon amplified region with the sequences of the same region of prototype strains existing in the GenBank database
In order to identify Enterovirus serotypes, pairwise comparison of the VP1 amplicon sequence was performed with the complete database of VP1 sequences of all known EV serotypes using the BLAST program (
Types of Adenoviruses were determined by comparing the sequences obtained from the hexon amplified region with the sequences of the same region of prototype strains existing in the GenBank database
9
DNA Digestion and Electrophoresis
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Digestion reaction was performed by incubating 1 μg of isolated DNA with 2.5 U of enzyme (ThermoScientific) in a final reaction volume of 20 μl at 37 °C for 2 h. The restriction fragments were separated by 1.5% agarose gel electrophoresis in TAE buffer for 2 h at 30 V and stained by the nucleic acid stain (SimplySafe™, Eurx).
10
Agarose Gel Electrophoresis Protocol
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Agarose gels were prepared at a concentration of 2% in 1× Tris-acetate-EDTA (TAE) and stained with SimplySafe (EURx, Gdańsk, Poland) nucleic acid stain. Electrophoresis was carried out in 1× TAE buffer for 1.5 h at 100 V. The molecular weight of obtained products was evaluated using GeneRulerTM 100 bp DNA Ladder Plus (Thermo Fisher Scientific, Waltham, MA, USA).
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