For northern blot analysis, total RNA was extracted from leaf tissues as described previously [37 (link)]. Total RNA (5 µg) was separated on 1% formaldehyde agarose gels, transferred to positively-charged nylon membranes (Roche Diagnostics, Mannheim, Germany), and probed with digoxigenin-labeled specific RNA probes for the RNA1 and RNA2 3′-untranslated region of TRV (6548–6789 nt of RNA1 and 1860–2101 nt of RNA2), and for the 3′-end region of PVX (5875–6403 nt of PVX cDNA sequence in pgR107 vector). For tissue blot hybridization, fresh cross-sections of four non-inoculated leaf petioles or stems were squash blotted on positively-charged nylon membranes (Roche Diagnostics, Mannheim, Germany), and blots were hybridized with the digoxigenin-labeled RNA probes mentioned above. The hybridization conditions used were those recommended by the manufacturer (DIG Application Manual for Filter Hybridization, Roche Diagnostics, Mannheim, Germany). Membranes were exposed to X-ray film (X-Omat AR, Kodak, Rochester, NY, USA) and developed following a conventional photographic process.
Positively charged nylon membrane
Manufactured by Roche
Sourced in Germany, United States, Switzerland, United Kingdom
The positively charged nylon membrane is a specialized laboratory equipment used in various biochemical and molecular biology applications. It is designed to facilitate the effective binding and immobilization of negatively charged biomolecules, such as nucleic acids and proteins, through electrostatic interactions. The membrane's positive charge enables efficient capture and retention of these target molecules, making it a valuable tool for techniques like blotting, hybridization, and antibody-based assays.
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Lab products found in correlation
Pcr dig probe synthesis kit, by Roche (27 mentions)
Cdp star, by Roche (19 mentions)
Dig northern starter kit, by Roche (14 mentions)
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Dig dna labeling and detection kit, by Roche (9 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (8 mentions)
Dig easy hyb, by Roche (8 mentions)
Anti dig antibody, by Roche (7 mentions)
Dig wash and block buffer set, by Roche (7 mentions)
Dig luminescent detection kit, by Roche (7 mentions)
Dig high prime dna labeling and detection starter kit 2, by Roche (6 mentions)
Imagequant las 500 imager, by GE Healthcare (5 mentions)
Cdp star substrate, by Roche (5 mentions)
Anti digoxigenin ap fab fragment, by Roche (5 mentions)
Blocking reagent, by Roche (5 mentions)
Chemidoc xrs system, by Bio-Rad (4 mentions)
Dig probe synthesis kit, by Roche (4 mentions)
Dig easy hyb solution, by Roche (4 mentions)
Alkaline phosphatase conjugated anti digoxigenin antibody, by Roche (4 mentions)
Dig gel shift kit, by Roche (4 mentions)
204 protocols using positively charged nylon membrane
1
Northern Blot Analysis of Plant Viruses
2
Quantifying Satellite DNA Proportions
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Genomic DNAs were digested with HaeIII (Fermentas), HpaII (Fermentas), MspI (Promega), PvuII (New England Biolabs) and TaqI (Roche). Restriction products were resolved by gel electrophoresis, transferred to positively charged nylon membranes (Roche), and hybridized overnight with the DIG-labelled SSUsat probe. To test homology in low, moderate and high stringency conditions, different experimental temperatures (60, 65 and 68 °C) were used. Signals were detected with alkaline phosphatase conjugated anti-digoxigenin antibodies (Roche) using CDP-Star (Roche) as substrate.
The proportions of the satellite present in the genomes of S. subtruncata, S. solida, M. stultorum and D. trunculus were estimated by dot blotting. Serial dilutions of genomic DNAs (10–250 ng) and cloned SSUsat (0.3–10 ng) were dot-blotted onto positively charged nylon membranes (Roche) and hybridized with SSUsat probes. Hybridisation signals were quantified using ImageJ (http://imagej.nih.gov/ij/ ).
The proportions of the satellite present in the genomes of S. subtruncata, S. solida, M. stultorum and D. trunculus were estimated by dot blotting. Serial dilutions of genomic DNAs (10–250 ng) and cloned SSUsat (0.3–10 ng) were dot-blotted onto positively charged nylon membranes (Roche) and hybridized with SSUsat probes. Hybridisation signals were quantified using ImageJ (
3
Isolation and Detection of miRNA
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Total RNAs were extracted from animal tissue or cultured cells using TRIzol reagent. The 18-to 24-nt miRNA fraction was isolated by polyacrylamide gel electrophoresis using a Gel Purification of miRNA from Total RNA Kit (Ambion) according to manufacturer's instructions. The same method was employed to isolate miRNAs from cells for HPLC analysis. Poly(A) + mRNAs were isolated using the Dynabeads mRNA Purification Kit (Ambion).
Purified miRNA samples were then separated on a denaturing 15% polyacrylamide gel and blotted onto positively charged nylon membranes (Roche). Total RNAs or purified mRNA samples were resolved in 1% agarose/formaldehyde gel and blotted onto positively charged nylon membranes (Roche). Following UV fixation, the membrane was washed with PBS followed by incubation in 1% BSA blocking solution for 1 hr at room temperature. The membranes were incubated in Oxidative Damage Markers Monoclonal Antibody (clone 15A3) (1:1,000; QED Bioscience) for 4 hr at room temperature and then incubated in a horseradish peroxidase conjugated goat anti-mouse secondary antibody (1:1,200; Sigma). The signals were detected with Pierce ECL Western Blotting Substrate (Pierce) according to the directions of the manufacturer and exposed on X-ray films (Kodak).
Purified miRNA samples were then separated on a denaturing 15% polyacrylamide gel and blotted onto positively charged nylon membranes (Roche). Total RNAs or purified mRNA samples were resolved in 1% agarose/formaldehyde gel and blotted onto positively charged nylon membranes (Roche). Following UV fixation, the membrane was washed with PBS followed by incubation in 1% BSA blocking solution for 1 hr at room temperature. The membranes were incubated in Oxidative Damage Markers Monoclonal Antibody (clone 15A3) (1:1,000; QED Bioscience) for 4 hr at room temperature and then incubated in a horseradish peroxidase conjugated goat anti-mouse secondary antibody (1:1,200; Sigma). The signals were detected with Pierce ECL Western Blotting Substrate (Pierce) according to the directions of the manufacturer and exposed on X-ray films (Kodak).
4
Detecting Red Complex Bacteria in Chronic Periodontitis
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Ten randomly selected subgingival plaque samples from VD patients with CP were assessed for the presences of red complex bacteria (RCB) using checkerboard DNA–DNA hybridization. NaOH (100 µl of 0.5 M) was added to 100 µl of subgingival plaque samples in TE buffer and vortexed. After boiling for 10 min, 800 µl of 5 M ammonium acetate was added to neutralize the samples. The DNA was then applied into the extended slots of a Minislot-30 apparatus (Immunetics, Cambridge, MA) with a positively charged nylon membrane (Boehringer Mannheim, Indianapolis, IN). The membrane had two lanes with DNA standards equivalent to 105–106 cells per target species tested. The membranes were hybridized with 20 digoxigenin labeled whole genome probes of species significant for the subgingival microbiota, including RCB (Table 2 ) (30 (link)). DNA from the type species was used as probes.
5
Ajoene-Induced RNA Expression in S. aureus
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Five µg of RNA extracted from 8325-4 cultures after 60′ (late exponential) and 240′ minutes (early stationary) of growth post ajoene treatment (see previous description) was loaded onto a 1% agarose gel and separated in 10 mM sodium phosphate buffer as described previously56 (link). RNA was transferred to a positively charged nylon membrane (Boehringer Mannheim) by capillary blotting. Hybridization was performed according to56 (link) using gene-specific probes that had been labeled with [32 P]dCTP using the Ready-to-Go DNA-labeling beads (Amersham Biosciences). Internal fragments of the hla or the rnalll gene were used as template in the labeling reactions. See Table S2 for primer sequences.
6
CRISPR Constructs Transfection and Northern Blot Analysis
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Equimolar amounts of CRISPR constructs (equivalent of 2 μg pX458) were transfected into 6 × 105 HEK293T cells in 6-well plates using Lipofectamine 2000 (Invitrogen). Cellular RNA was extracted 2 days post-transfection, and 5 μg total RNA was separated in a 15% denaturing polyacrylamide gel (Precast Novex TBE Gel, Life Technologies) and then electro-transferred to a positively charged nylon membrane (Boehringer Mannheim). Locked nucleic acid (LNA) oligonucleotides pLuc1 or pSca (Table S1 ) were 5′ end-labeled with the KinaseMax Kit (Ambion) in the presence of 1 μL [γ-32P]-ATP (0.37 MBq/μL, PerkinElmer). The membrane was incubated overnight at 42°C with the labeled LNA oligonucleotides. The blot was washed twice for 5 min at 42°C with 2 × SSC/0.1% SDS and twice for 5 min at 42°C with 0.1 × SSC/0.1% SDS. The signals were exposed by the Typhoon FLA 9500 (GE Healthcare), and densitometric analyses were performed using ImageJ software. Two independent transfections and Northern blots were performed.
7
Measuring Telomere Length via Restriction Fragment Analysis
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Telomere restriction fragments were analyzed using the TeloTAGGG Telomere Length Assay kit (Roche). Briefly, cells were harvested by trypsinization, washed in PBS and collected by centrifugation at 400 g for 4 min. Genomic DNA was isolated using DNeasy Blood and Tissue Kit (Qiagen), digested with HinfI and RsaI restriction enzymes (New England Biolabs) and separated by gel electrophoresis either on 0.8% agarose gels at 50V overnight in 1X TBE buffer or (to resolve elongated telomeres at later time points) on 1% megabase agarose gels (Bio-Rad) using a CHEF DRII equipment (Bio-Rad) under the following conditions: 120° field angle, 5 to 30 s switch times, 5 V/cm and 14°C for 14 hr in 1X TAE. Following the resolution of DNA fragments, DNA was transferred to a positively charged nylon membrane (Roche) by Southern blotting and hybridized with a digoxigenin-labelled telomeric probe. Membranes were exposed to X-ray film (Carestream) and developed in X-OMAT 2000 Processor (Kodak). Mean telomere lengths were calculated as described in Kimura et al. (2010) (link).
8
Northern Blot of L1 Consensus and RRACH Mutation
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Northern blot was performed using DIG Northern Starter Kit (Roche, 12039672910) following the manufacturer’s instructions. In brief, RNA was separated with 1.2% formaldehyde agarose gel and transferred to a positively charged nylon membrane (Roche) by capillary transfer overnight with standard 2× SSC solution. Membrane was UV crosslinked with 400 millijoule (mJ)/cm2. Membrane was hybridized with denatured DIG-labeled RNA probe overnight at 68 °C. Strand specific DIG labeled RNA probe was generated with MEGAscript™ T7 Kit (Thermo Fisher). DNA template for probe synthesis was generated by primers specifically targeting the first 100 bp of reporter L1 region, which is shared by both L1 consensus and RRACH mutation reporter (Sequences see Supplementary information, Table S4 ). T7 promoter sequence was added to the beginning of the reverse primer. Hybridized nylon membrane was washed with four rounds of stringent washes (with 0.1% SDS). Membrane was then blocked, washed and imaged following manufacturer’s instructions.
9
Genomic DNA Extraction and Hybridization
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Genomic DNA was extracted from leaves of individual seedlings using the Nucleon Phytopure extraction kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. After endonuclease digestion and electrophoresis on a 1% agarose gel, DNA fragments were transferred to a positively charged nylon membrane (Roche). Probes were prepared using a PCR DIG probe synthesis kit (Roche). Hybridization was performed according to the DIG Application Manual (Roche). Hybridization was at 42°C and washing was performed under high-stringency conditions at 68°C.
10
Quantitative Dot Blot Analysis of DNA Targets
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For genomic DNA, 50, 100, and 200 ng were spotted onto positively charged nylon membrane (Roche). PCR products corresponding to CL25m1, CL16m1, and CL16m3/32m1 fragments were spotted in the amounts of 0.5, 1, 2, 4, and 8 ng. Hybridization was done in 250 mM phosphate buffer pH 7.2, 1 mM EDTA pH 8, 20% SDS, and 0.5% Blocking Reagent (Roche) with 50 ng of biotin-labeled probes at 65 °C with agitation overnight. Posthybridization washes were done in 20 mM Na2HPO4, 1 mM EDTA, and 1% SDS 3 × 20 min at 62 °C. Detection was carried out using streptavidin-AP-conjugate (1:5,000, Roche) followed by chemiluminescence with AP substrate CDP-Star (1:50, Roche). Dot blot intensities were compared using ImageJ with measurement of mean gray values that were inverted and normalized for background.
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