Aliquots of EDTA blood samples were mixed with RNALater (Ambion, Foster City, CA, USA) for storage. Subsequently, blood cells were washed in PBS, and RNA was purified (PureLink RNA Mini Kit, Invitrogen, Carlsbad, CA, USA) per the manufacturer’s instructions. Purified RNA (43.5 ng from proband, 12 ng from a healthy control cat) was subjected to reverse transcription-PCR (RT-PCR) using a SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (ThermoFisher Scientific, Waltham, MA, USA). The RNA was reverse transcribed at 50°C for 30 min, and the cDNA was amplified using 10 pmol primer sets 1–7 (for primer sequences see Table S1 ) with initial denaturation at 94°C for 2 min, 40 cycles at 94°C 15 s, 55°C 30 s, 68°C 60 s, and a final extension of 5 min at 68°C. The PCR products were electrophoresed on 1.2% Seakem LE agarose gels (Lonza, Walkersville, MD, USA), and bands were individually excised, purified (QIAquick Gel Extraction Kit, Qiagen, Valencia, CA, USA) and sequenced by Sanger sequencing (CCR Genomics Core of the National Cancer Institute, Bethesda, MD, USA).
Seakem le agarose gel
Manufactured by Lonza
Sourced in United States, Switzerland
SeaKem LE agarose gel is a laboratory equipment product used for electrophoresis applications. It is a purified agarose material that forms a gel matrix to separate and analyze DNA, RNA, or protein samples.
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Lab products found in correlation
Qiaquick gel extraction kit, by Qiagen (5 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (5 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Puc57 plasmid vector, by Bio Basic (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Superscript 3 one step rt pcr system, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Gotaq dna polymerase, by Promega (1 mentions)
Cloneamp hifi pcr premix, by Takara Bio (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Sanger sequencing, by Eurofins (1 mentions)
Thermocycler, by Bio-Rad (1 mentions)
Gotaq green master mix, by Promega (1 mentions)
Genelute bacterial genomic dna kit, by Merck Group (1 mentions)
Genematrix stool dna purification kit, by EURx (1 mentions)
Ppi 200 programmable power inverter, by Bio-Rad (1 mentions)
25 protocols using seakem le agarose gel
1
RNA Purification and Sequencing from Blood
2
Cloning and Transformation Protocols
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E. coli DH5α-competent cells were prepared according to the calcium chloride protocol, as described previously [62 (link)]. All standard molecular cloning procedures were carried out as described in Sambrook and Russell (2001) [63 ] or according to manuals provided by producers. Genomic DNA from B. subtilis 168, B. methanolicus MGA3 and E. coli MG1655 was isolated [64 (link)]. PCR products were amplified using Cloneamp HiFi PCR Premix (Takara) and purified using a QIAquick PCR Purification kit from Qiagen. DNA fragments were separated using 0.8% SeaKem LE agarose gels (Lonza) and isolated using a QIAquick Gel Extraction Kit (Qiagen). DNA fragments were joined by the means the isothermal DNA assembly [65 (link)]. Table 3 presents the list of DNA fragments that were used for plasmid construction this study. Colony PCR was performed using GoTaq DNA Polymerase (Promega). The sequences of cloned DNA fragments were confirmed by Sanger sequencing (Eurofins). B. methanolicus MGA3 and M168-20 were made electrocompetent and transformed by electroporation as described by Jakobsen et al. (2006) with modifications [56 (link),66 (link)].
3
ERIC-PCR Genomic Profiling of E. coli and K. pneumoniae
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The genomic DNA of E. coli and K. pneumoniae was prepared by boiling the isolates and subsequently used as templates. Bacterial genomic DNA was extracted by emulsifying a loop full of bacteria from a Luria-Bertani agar plate (Fisher Scientific) into 300 µl of sterile, molecular biology-grade water. The tube was then incubated at 95°C in a heat block for 15 min and centrifuged at 13,000 rpm for 10 min. Two microlitres of the supernatant were used as a DNA template. ERIC-PCR was performed using a thermocycler (Bio-Rad) and ERIC primers; forward: 5'-ATG TAA GCT CCT GGG GAT TCAC-3' and reverse: 5'-AAG TAA GTG ACT GGG GTG AGC G3' [14 (link)]. The amplification was performed by adding a mixture (25 µL per reaction) of 12.5 µL GoTaq green master mix (M7122, Promega), 2 µL primers of each primer, 9.5 µL nuclease-free water, and 1 µL of DNA template. The PCR protocol consisted of an initial denaturation (94°C for 5 min) followed by 40 cycles of denaturation (95°C for 1 min), annealing (51°C for 1 min) followed by 65°C for 8 min, and a final extension (72 °C for 10 min) [14 (link)]. The PCR products were loaded onto a 1.5% SeaKem LE agarose gels (Lonza), with 1 KB and 100 Bp DNA ladder (MOLEQULE-ON) separated at a constant voltage of 90 V for 1.5 hours, and the banding patterns were visualized under ultraviolet radiation.
4
Extracting and Purifying E. coli rDNA
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The total rDNA was extracted from faecal and lagoon samples with the GeneMATRIX Stool DNA Purification Kit (E3575, EURx Ltd, Gdańsk, Poland). The rDNA concentration and purity were determined with NanoDrop 1000 (Thermo Fisher Scientific Inc., Waltham, MA, USA) by migration in 0.8% SeaKem® LE Agarose gels (50004L, Lonza Group Ltd, Basel, Switzerland) in a 1× TBE buffer. The 16S rDNA from the confirmed E. coli was isolated with the GenEluteTM Bacterial Genomic DNA Kit (NA2120, Merck (Sigma-Aldrich, St. Louis, MO, USA).
5
Antibiotic Resistance Profiling of E. coli
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The E. coli strains modified in this study are listed in Table 1 . Bacteria were grown in liquid Luria–Bertani medium (LB), or on LB plates containing 1.5% agar. Components of the media were obtained from Molar Chemicals Kft., Halásztelek, Hungary. Antibiotics were obtained from Sigma Aldrich (St. Louis, MO, USA) and were used in the following concentrations: chloramphenicol (Cm): 25 μg/mL; ampicillin (Ap): 100 μg/mL; kanamycin (Km): 25 μg/mL; spectinomycin (Sp): 50 μg/mL; anhydrotetracycline (aTc): 50 ng/mL. Antibiotic-gradient plates were made applying the protocol of Szybalski and Bryson [34 (link)], but using 60× higher Sp and 20× higher Km concentrations in the top layer, compared to the values listed above. Plasmid preparations were made using the Zippy Plasmid Mini Prep Kit (Zymo Research Ltd., Orange County, CA, USA). The horizontal electrophoresis of DNA was carried out using 1% Seakem LE agarose gels (Lonza, Basel, Switzerland). All cloning and molecular biology experiments were carried out according to established protocols, unless stated otherwise [35 ].
6
Plasmid DNA Electrophoresis Separation
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Plasmid DNA (500 ng) was electrophoresed on a 0.7% SeaKem LE Agarose gel (Lonza, Basel, Switzerland) with 0.5× TBE buffer. DNA was initially electrophoresed without inversion at 100 V for 15 min, followed by 24 hr of inversion with recirculation of buffer at 4°C. The reverse‐pulse electrical field was supplied by a PPI‐200 Programmable Power Inverter (MJ Research, Watertown, MA) on Program 3.
7
Equine VEGFα Expression Quantification
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The horse VEGFα expression level was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) amplification. The primers were designed using the PRIMER3 software (MIT, Cambridge, MA, USA, http://frodo.wi.mit.edu/primer3/ ) with an expected product size of 184 bp. To detect equine VEGFα mRNA, following primers were used: forward 5′-CTA CCT CCA CCA TGC CAA GT-3′ and reverse 5′-CAC ACA GGA TGG CTT GAA GA-3′. The RT-PCR conditions were as follows: an initial step of 94°C for 10 min, 35 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, and a final step of 72°C for 10 min. RT-PCR products were analyzed with gel electrophoresis on a 2.0% SeaKem LE agarose gel (Lonza, NJ, USA). Equine GAPDH gene was used as a normalizer for RT-PCR.
8
Gut Microbiome Profiling in Piglets
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Samples from two representative animals were selected from each farm: one piglet with NNPD and one piglet without NNPD, giving a total of 16 samples to be sequenced (when samples from both the ileum and colon of each animal were included). Piglets were chosen to be approximately the same age (5 or 6 days old). Concentrations of the respective PCR amplicons for each animal were determined using the Agilent 1000 chip (Agilent Technologies, Waldbronn, Germany). Amplicons were subsequently pooled in equal concentrations and size-separated by running the amplicons for 86 min, 90 V, in a 0.7% Seakem® LE Agarose gel (Lonza Rockland, Rockland, ME, USA) followed by incubation for 30 min in 0.0004% ethidium bromide for staining. By means of UV radiation from the Bio-Rad Universal hood II (Segrate, Milan, Italy) gel bands were visualized and bands spanning the size range of the primer products (200–900 base pairs) were excised. Finally, the Qiaquick Gel Extraction Kit (Qiagen) was used to purify DNA from the gel. This pool of 1615.7 ng DNA (260/280 nm-ratio: 1.97) derived from ileal and colonic luminal contents of 8 different animals was sequenced on a half PicoTiterPlate™ by a 454 GS FLX Titanium Sequencer (Roche) via LGC Genomics (GmbH, Berlin, Germany).
9
Gel Electrophoresis of VNTR Regions
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The VNTR regions were analyzed using gel electrophoresis (1 V/cm) in 1× TAE buffer through a 0.7–2.0% agarose gel. The electrophoresis conditions were set differently depending on the repeat unit and length of the PCR product for each VNTR region. For MS1, a 2% SeaKem ® LE agarose gel (Lonza, Rockland, ME, USA) was run at 120 V for 6 h. MS2 was run on a 1.5% LE agarose gel at 80 V for 14 h, and MS4 was run on a 2% LE agarose gel at 120 V for 4 h. A 100 bp DNA ladder (ELPIS-Biotech, Daejeon, Republic of Korea) was used as a marker for the electrophoresis of these three regions. The length of the repeat unit for MS3 was 29 bp. Since the length of the PCR product was about 3 kb, the PCR product was run on a 0.7% LE agarose gel at 60 V for 20 h. In this case, a 1 kb DNA ladder (Invitrogen, Carlsbad, CA, USA) was used as a marker.
10
Microsatellite Analysis of Olive Accessions
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A set of 11 microsatellite markers [simple sequence repeat (SSR)] was selected as the most effective in differentiating the olive accessions (Table S1 ) (Boucheffa et al., 2017 (link)). PCR reactions were performed in a C1000 TouchTM Thermal Cycler (Bio-Rad, Hercules, CA, USA) following the protocol described in Montemurro et al. (2015) (link). In order to verify PCR efficiency, PCR products for each of the 11 SSR markers were randomly checked by electrophoresis on 2.5% SeaKem LE Agarose gel (Lonza, Visp, Switzerland). The amplification products were detected by the automatic sequencer ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), and the sample analyses were carried out using the GeneMapper genotyping software v3.7 (Applied Biosystems, Foster City, CA, USA). The internal molecular weight standard was GeneScanTM 500 ROX dye Size Standard (Applied Biosystems, Foster City, CA, USA).
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