The copy number of the inserted BAC clone in the BAC-TG mice was determined by performing Southern blotting. Genomic DNA, which was prepared from the lung, was treated with EcoRV (New England Biolabs Japan, Tokyo, Japan). The DNA was electrophoresed on 0.7% agarose gels and transferred onto GeneScreen Plus Hybridization Transfer Membranes (PerkinElmer, Waltham, MA, USA), and then blotted and detected using DIG-labelled DNA probes synthesised by using a DIG PCR Synthesis Kit (Roche Diagnostics Japan, Tokyo, Japan) in accordance with the manufacturer’s protocol.
Genescreen plus hybridization transfer membrane
Manufactured by PerkinElmer
Sourced in United States
The GeneScreen Plus Hybridization Transfer Membrane is a nylon-based membrane used for the immobilization and transfer of nucleic acid samples in molecular biology applications. It is designed to facilitate efficient hybridization and detection of target sequences.
Automatically generated - may contain errors
Lab products found in correlation
T4 polynucleotide kinase, by New England Biolabs (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Ecorv, by New England Biolabs (2 mentions)
Gamma 32p atp, by PerkinElmer (2 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (2 mentions)
Pcr dig synthesis kit, by Roche (1 mentions)
Cyclone phosphor imager, by PerkinElmer (1 mentions)
Random priming kit, by Promega (1 mentions)
Northernmax hybridization buffer, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna kit, by Qiagen (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Hyblot es autoradiography film, by Thomas Scientific (1 mentions)
Neb2 buffer, by New England Biolabs (1 mentions)
Criterion 5 tbe precast gels, by Bio-Rad (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Chef dr 2 system, by Bio-Rad (1 mentions)
Amersham gene images alkphos direct labelling and detection system, by GE Healthcare (1 mentions)
Spei hf, by New England Biolabs (1 mentions)
Dig easy hybtm, by Roche (1 mentions)
17 protocols using genescreen plus hybridization transfer membrane
1
Southern Blot Analysis of BAC-TG Mice
2
Northern Analysis of U1 snRNA
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Northern analysis of U1 snRNA was carried out as follows. Total RNA was prepared and separated on 8% polyacrylamide-7 M urea gels, then transferred to GeneScreen Plus Hybridization Transfer Membranes (PerkinElmer) using a semi-dry transfer apparatus. Northern hybridization was performed using the 32P 5′-end-labelled oligonucleotide probes listed in Supplementary Table 1 . U6 snRNA was used as loading control. The quantification was performed using OptiQuantTM software with a Cyclone Phosphor Imager (PerkinElmer).
3
High-Resolution Northern Blotting for Small RNAs
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Northern blotting for small RNAs followed previously described probes and procedures (Okamura et al. 2007 (link)). To obtain single-nucleotide resolution of 55–80 nt pre-miRNAs, 8–15 mg of total RNA was loaded on 12% polyacrylamide–urea sequencing gels (40 cm long) as previously described (Bortolamiol-Becet et al. 2015 (link)). Briefly, gels were pre-run in 1× TBE for 30 min at 50 mA, and samples were run at 20 mA for 20 min, then at 50 mA for 5–6 h. We transferred them to GeneScreen Plus Hybridization Transfer Membranes (PerkinElmer) in 0.5× TBE for an hour at 300 mA on a semi-dry transfer system and then prehybridized and hybridized them using standard procedures (Okamura et al. 2007 (link)).
4
Viral Infection Assay and Analysis
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HEK-293 cells, MRC-5 cells, Vero cells or L2-5 cells were infected with viruses indicated in the figures at a multiplicity of infection (MOI) of 5. Total protein or RNAs were prepared at different time points post inoculation. Western blot was performed using the antibodies described above. For RT-PCR, total RNAs were extracted using All-Prep DNA/RNA kits (Qiagen). The primer sequences are listed in S3 Table . The RT-PCR bands shown in the figures that correspond to novel splice junctions were further confirmed by Topo cloning and sequencing. HEK-293 cells were transfected with plasmids indicated in Fig 6D using Lipofectamine 2000 (Invitrogen). Total protein or RNAs were prepared 24 hours post transfection. For Northern blots, total RNAs were prepared from HEK-293 cells or Vero cells infected with HSV-1 KOS strain or ICP27 mutants by TRIzol (Invitrogen). Approximately 30 μg of total RNAs were separated in a formaldehyde denaturing 1.2% agarose gel (Life Technologies). After transfer to GeneScreen Plus hybridization transfer membrane (Perkin-Elmer), the membrane was incubated in NorthernMax hybridization buffer (ThermoFisher Scientific) at 58°C overnight with an HSV-1 ICP34.5-specific probe labeled with [α-32P] dCTP using a random priming kit (Promega).
5
Telomere Length Analysis Using Bal31 Exonuclease
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The assay was performed with Bal31 exonuclease (Takara Bio, Shiga, Japan) according to the manufacturer's instructions. Briefly, 1 µg of total DNA was adjusted to 250 µl with the 1× Bal31 nuclease buffer provided by the manufacturer. Samples (62 µl) were removed at regular intervals depending on the strain. The reactions were terminated by adding EGTA (Sigma) to a final concentration of 10 mM. The samples were ethanol precipitated, and the DNA was digested with PstI (Invitrogen) and electrophoresed on 1% agarose gels. The samples were then transferred to nylon membranes (GeneScreen Plus, Hybridization Transfer Membrane, Perkin Elmer Inc., Alameda, CA, USA) and hybridized to (TTAGGG)n, UT4-a, both, or rRNA gene sequences as a probe.
6
Klf13 Binds to IL-4 Promoter
7
Northern Blot Analysis of Small RNA Expression
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Total RNA was isolated from ASO-treated primary neurons (10 μM, 72 h) by TRIzol (Life Technologies) according to the manufacturer’s protocol. 3 μg total RNA was separated on an 8% polyacrylamide-7M urea gel, and then transferred by semi-dry transfer (12 V, 30 min) to GeneScreen plus hybridization transfer membrane (Perkin Elmer). The Northern probes were 5′ end labelled with ATP Gamma 32P (Perkin Elmer) using T4 polynucleotide kinase (New England Biolabs), and then hybridized to the membrane at 42°C for 30 min. After washing membrane in wash buffer (2X SSC containing 0.1% SDS), the membrane was exposed to a PhosphorImager and quantified. The oligonucleotide probe sequences used were Snord116 5′-TTCCGATGAGAGTGGCGGTACAGA-3′ and 5.8S rRNA 5′-TCCTGCAATTCACATTAATTCTCGCAGCTAGC-3′.
8
Characterization of PATZ1 Transcription Factor Binding
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HepG2 cell nuclear extract with ectopically expressed 3xFLAG-PATZ1 was extracted using NucBuster Protein Extraction kit (Novagen) and quantified with Qubit 2.0 Fluorometer (Life Technologies). The oligonucleotides were minimized to be around 20 bp in length with several nucleotides flanking both sides of the motif sequence for PATZ1. A list of probes used in this assay can be found in Supplementary Table S3 . Both biotinylated and non-biotinylated probes were annealed with reverse complementary nucleotide sequences by NEB2 buffer (NEB). A total of 6 μg nuclear extracts were incubated with 200 fmol of biotinylated double-stranded probes for 40 min on ice in 10 mM Tris–HCl, 80 mM KCl, 1 mM DTT, 1 μg of Poly (dA-dT), 7.5% glycerol, 0.063% NP-40 and 2 mM MgCl2. For competition assay, extra amounts of unlabeled probes were added to the binding reaction and further incubated at room temperature for 20 min. Samples were separated in Criterion 5% TBE Precast Gels (Bio-rad), and electro-transferred into a Genescreen plus hybridization transfer membrane (Perkin Elmer). DNA–protein complexes were cross-linked using UV-light and detected by LightShift Chemiluminescent EMSA Kit (Life Technologies).
9
Genomic DNA Digestion and Southern Blot
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Extracted DNA was digested with ApaI, XbaI or XhoI as indicated in the figure legend. Four μg DNA were digested for 8 h at 37°C prior to electrophoretic size separation of fragments in a 0.8% agarose gel. The DNA was blotted overnight onto a GeneScreen Plus Hybridization Transfer Membrane (PerkinElmer). The filters were hybridized with a 32P-dCTP labeled unit-length MnPV DNA as previously described [16 (link)].
10
Mitochondrial DNA Verification in Kudoa Infection
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To verify that the obtained genome sequence was derived from the mitochondrial chromosome and not from the nuclear copies of mitochondrial DNA (in nuclear chromosomes) or from fish mitochondria, we performed Southern blotting. We performed pulsed field electrophoresis of K. septempunctata isolate 0904 total DNA and Kudoa-free Paralichthys olivaceus total DNA (control) on a 1% agarose gel in 0.5x TBE. Using CHEF-DR II system (Bio-Rad), the electrophoresis was performed under 6 V/cm, 120° angle in two blocks: the run time was 3 h with 0.1–1 sec switch time ramp, followed by 11 h with 1–2 sec switch time ramp. DNA was transferred from the gel to a GeneScreen Plus Hybridization Transfer Membrane (PerkinElmer) by the alkaline transfer method. DNA probes were labeled using Amersham Gene Images AlkPhos Direct Labelling and Detection System (GE Healthcare) and hybridized to the DNA on the membrane. The probes were PCR products amplifying the region at 10194–11194 bp (within cox1 gene) or the region at 17170–17760 bp (no gene) of K. septempunctata isolate 0904 genome.
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