RNA samples were separated on 12% polyacrylamide/8M urea gel. Soak Hybond‐N+ membrane (Amersham Pharmacia) in ddH2O for a few seconds and in transfer buffer (0.5× TBE) for 20 minutes and soak two pieces of Whatman paper in 0.5× TBE. Separated RNA in gel was electro‐blotted onto Hybond‐N+ membrane (Amersham Pharmacia). After UV cross‐linking and air‐drying, blotted membrane was prehybridized with hybridization buffer at 42°C for 60 min and then hybridized with biotin‐labelled antisense miR24‐2 probe and incubated at 42°C for overnight. The membrane was washed 4 times at 42°C with 2× SSC and 0.5% SDS and then Western blotting with anti‐biotin according to our pervious protocol.35
Hybond n membrane
Manufactured by Cytiva
Sourced in United Kingdom, United States, Sweden, Germany, China, Canada
Hybond-N+ membrane is a nylon-based membrane used for DNA and RNA transfer and immobilization in molecular biology applications. It provides a stable and efficient platform for blotting and hybridization experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (21 mentions)
Trizol, by Thermo Fisher Scientific (19 mentions)
Dig high prime dna labeling and detection starter kit 2, by Roche (16 mentions)
Pcr dig probe synthesis kit, by Roche (15 mentions)
Tri reagent, by Merck Group (14 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
Dig high prime dna labeling and detection starter kit 1, by Roche (6 mentions)
Uv crosslinker, by Avantor (5 mentions)
Cdp star, by Roche (5 mentions)
Dig high primer dna labeling and detection starter kit 1, by Roche (5 mentions)
Dig dna labeling and detection kit, by Roche (5 mentions)
Phosphorimager, by GE Healthcare (5 mentions)
Perfecthyb plus hybridization buffer, by Merck Group (5 mentions)
Ultrahyb oligo hybridization buffer, by Thermo Fisher Scientific (5 mentions)
Dig high prime kit, by Roche (4 mentions)
Ecl western blotting detection kit, by Thermo Fisher Scientific (4 mentions)
Dneasy blood and tissue kit, by Qiagen (4 mentions)
T4 polynucleotide kinase, by New England Biolabs (4 mentions)
Chemidoc touch imaging system, by Bio-Rad (4 mentions)
Dig northern starter kit, by Roche (4 mentions)
374 protocols using hybond n membrane
1
Northern Blot Analysis of miRNA-24-2
2
Genomic Southern Blot of C. neogracile
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Purified gDNA of C. neogracile (10 μg) was digested with EcoRV, KpnI, and XbaI, separated on a 0.8% agarose gel, and transferred to a Hybond™-N+ membrane (Amersham, Dayton, TN, USA). Genomic Southern blot was conducted by standard protocols using the radiolabeled Cn-isoAFP gene sequence as a probe [55 ].
Total RNA of C. neogracile was extracted using a Plant RNeasy mini kit (Qiagen, Hilden, Germany). RNA (10 μg) was incubated at 65 °C for 5 min for denaturation and loaded on an agarose gel (1.2% agarose, 10% 10X MOPS, and 4.75% formaldehyde). The RNA was then transferred to a Hybond™-N+ membrane (Amersham, Dayton, TN, USA) and UV-cross linked for 10 min. The Cn-isoAFP probe was amplified by 5′-upstream primers specific for Cn-isoAFP (Table S1 , #9 and #10). Probes were labeled with 32P-dCTP and hybridized in hybridization buffer (5X SSC, 5X Denhardt’s reagent, 0.5% SDS, 100 μg/mL herring sperm DNA) at 62 °C. The membrane was washed twice with wash buffer (2X SSPE and 0.1% SDS) for 25 min. The washed membrane was exposed to an X-ray film and developed.
Total RNA of C. neogracile was extracted using a Plant RNeasy mini kit (Qiagen, Hilden, Germany). RNA (10 μg) was incubated at 65 °C for 5 min for denaturation and loaded on an agarose gel (1.2% agarose, 10% 10X MOPS, and 4.75% formaldehyde). The RNA was then transferred to a Hybond™-N+ membrane (Amersham, Dayton, TN, USA) and UV-cross linked for 10 min. The Cn-isoAFP probe was amplified by 5′-upstream primers specific for Cn-isoAFP (
3
RNA Separation and Detection Protocols
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Separation of large RNA molecules: 4 μg total RNA was loaded in each lane of a 1.5% agarose gel with formamide and MOPS buffer. After electrophoresis, the RNA was transferred to a Hybond-N membrane (Amersham) by capillary blotting, UV crosslinked, and the marker as well as ribosomal RNA were visualised with methylene blue
Separation of small RNA molecules: 5 μg total RNA was loaded in each lane of an 8% acryl-amide gel with 8M urea. After electrophoresis, the RNA was transferred to a Hybond-N membrane (Amersham) by electroblotting, UV crosslinked, and the marker was visualised with methylene blue.
The 32P labelled synthetic DNA probes were detected with a Typhoon FLA7000 phosphoimager (General Electric). The membrane was stripped and re-used multiple times, each time verifying the stripping by phosphoimager (see Supplemental Experimental Procedures).
Separation of small RNA molecules: 5 μg total RNA was loaded in each lane of an 8% acryl-amide gel with 8M urea. After electrophoresis, the RNA was transferred to a Hybond-N membrane (Amersham) by electroblotting, UV crosslinked, and the marker was visualised with methylene blue.
The 32P labelled synthetic DNA probes were detected with a Typhoon FLA7000 phosphoimager (General Electric). The membrane was stripped and re-used multiple times, each time verifying the stripping by phosphoimager (see Supplemental Experimental Procedures).
4
Standard Molecular Techniques for PCR and Blot Analysis
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Standard molecular techniques were as described previously [16] (link). PCR reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science) or DyNAzyme polymerase II (Finnzymes; Espoo, Finland), according to the manufacturers' instructions. Southern blot analysis was carried out using Hybond-N+ membranes (Amersham Biosciences); northern blot analysis was performed as detailed [20] (link) using Hybond-N membranes (Amersham Biosciences). Oligonucleotides used in this study are detailed in Table 2 .
5
Jatropha Endosperm Total RNA Isolation
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Total RNA was isolated from jatropha endosperm using methods described previously [32 (link)]. About 10 μg total RNA was fractionated on a 1.2 % formaldehyde agarose gel and blotted onto a HybondTM N+ membrane (Amersham Biosciences, Little Chalfont, Buchinghamshire, UK). The DNA probe for the C1 gene (C1 probe) for northern blot analysis was the PCR products amplified from jatropha genome with primers C1SP-F and C1SP-R (Table 1 ). The northern blot hybridization and the labelling of the C1 probe were similar to the methods described for the Southern blot analysis.
6
Northern Blot Analysis of Fungal RNA
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RNA was isolated from the mycelium using TRI Reagent (Sigma-Aldrich Inc., St. Louis, MO, USA) as per manufacturer’s protocol. Ten micrograms of total RNA were separated using a 1.2% agarose gel with 1.85% (w/v) formaldehyde. The gel was blotted onto a HybondTM-N+ membrane (Amersham Biosciences, Slough, UK). The detection by hybridization was performed using digoxigenin-labelled probes amplified by PCR. Primers used for the amplification of the Northern blot hybridization probes are listed in Supplementary Table S3 .
7
Capped RNA Translation and Ribosome Assembly
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The capped RNAs from pluc, pHBV-TR-luc, and pSARS2-5′UTR-luc were generated by in vitro transcription using a mMESSAGE mMACHINE® Kit (AM1344; Invitrogen). Biotin-11-UTP (AM8450; Invitrogen) was added to the reaction mixture for producing biotin-labeled capped RNAs. The in vitro translation and ribosome assembly assays were performed as previously described (Locker et al., 2007 (link)). Briefly, the template RNAs and recombinant human RBM24 protein (rhRBM24) (Cao et al., 2018 (link)) or a non-specific control protein (BSA), were incubated in an RRL (L4960; Promega)-based translation reaction system. The luciferase activity assay was subsequently performed using a Steady-Glo® luciferase assay system (E2520; Promega) (Cao et al., 2014 (link)).
For the ribosome assembly assay, 1 μg of the biotin-labeled 5ʹ-UTRs of SARS-CoV-2 RNA and 12.5 pmol of rhRBM24 or BSA were added to an RRL-based ribosome assembly mixture. The mixtures were incubated for 15 min at 30 °C and subjected to 10–40% sucrose density gradient ultracentrifugation. The gradients were fractionated into 21 fractions and blotted onto an Amersham Hybond™-N+ membrane with a Whatman® Minifold® I 96 well dot-blot array system. The membrane was crosslinked with a HL-2000 Hybrilinker, and the blotted biotinylated-RNA was detected with Streptavidin-HRP (CT353; U-Cytech).
For the ribosome assembly assay, 1 μg of the biotin-labeled 5ʹ-UTRs of SARS-CoV-2 RNA and 12.5 pmol of rhRBM24 or BSA were added to an RRL-based ribosome assembly mixture. The mixtures were incubated for 15 min at 30 °C and subjected to 10–40% sucrose density gradient ultracentrifugation. The gradients were fractionated into 21 fractions and blotted onto an Amersham Hybond™-N+ membrane with a Whatman® Minifold® I 96 well dot-blot array system. The membrane was crosslinked with a HL-2000 Hybrilinker, and the blotted biotinylated-RNA was detected with Streptavidin-HRP (CT353; U-Cytech).
8
Optimizing RNA Annealing and Binding
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The IVT RNA was subjected to different annealing protocols. It was heated in either TE or TE/100 mM KCl at 95°C for 10 min. Then it was either put on ice for ≥2 min, kept at room temperature or cooled slowly to room temperature. These samples then had either protocol #1 buffer added so the final concentration was 50 mM Tris-HCl pH 7.5 at 25°C, 100 mM KCl, 5 mM MgCl2, 0.5 mM ZnCl2, 0.1 mM CaCl2, 2 mM 2-mercaptoethanol, 0.1 mg/mL bovine serum albumin, 0.1 mg/mL fragmented yeast tRNA (Sigma cat # R5636), 5% v/v glycerol or protocol #2 buffer added so the final concentration was 50 mM Tris pH 8.0, 50 mM NaCl, 0.2% CHAPS, 2 mM EDTA, 0.5% BSA, 5% glycerol, 0.1 mg/mL fragmented yeast tRNA (Sigma cat # R5636). This was then added to protein, incubated at 37°C for 20 min. After the incubation, the tubes were placed in an aluminum block on ice. As above, the sample was then loaded onto a 10 × 15 cm, 1× TBE/0.8% GTG agarose gel. The gel was run for 90 min at 66 V. When finished running the gel was placed on Hybond N+ membrane and Whatman 3 mm chromatography paper and dried down for 1 h at 80°C. The dried gels were exposed to phosphorimaging plates and signal acquisition was performed using a Typhoon Trio phosphorimager (GE Healthcare).
9
Extraction and Analysis of Fungal and Plant DNA
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Total DNAs from fungi were extracted following the method of Raeder and Broda (Raeder and Broda, 1985 (link)), using mycelium collected from a PDB culture incubated at 28°C and 200 rpm for 2 days. DNA isolation from tomato roots was performed with the cetyltrimethylammonium bromide (CTAB) extraction method (Dellaporta et al., 1983 (link)). Total RNA was extracted using TRIZOL® reagent (Invitrogen Life Technologies, Carlsbad), following the instructions of the manufacturer.
For Southern analyses, 10 μg of genomic DNA was SacI-digested, electrophoresed on a 0.7% agarose gel and transferred to a Hybond-N+ membrane (Amersham Biosciences AB, Uppsala, Sweden). The phleomycin gene was labeled using the DIG High Prime kit (Roche, Penzberg, Germany), following the manufacturer's instructions, and used as probe. Hybridization, washes and detection were carried out as previously described (Tijerino et al., 2011 (link)).
For Southern analyses, 10 μg of genomic DNA was SacI-digested, electrophoresed on a 0.7% agarose gel and transferred to a Hybond-N+ membrane (Amersham Biosciences AB, Uppsala, Sweden). The phleomycin gene was labeled using the DIG High Prime kit (Roche, Penzberg, Germany), following the manufacturer's instructions, and used as probe. Hybridization, washes and detection were carried out as previously described (Tijerino et al., 2011 (link)).
10
Screening Colonies for Oxidase Activity
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Ligation reactions were
transformed into E. coli competent cells (T7 express,
New England Biolabs) and spread onto an LB agar with 100 μg
mL–1 ampicillin covered with a Hybond-N membrane
(Amersham Biosciences). Following incubation overnight at 30 °C,
the membrane containing single colonies was transferred to an LB agar
plate (100 μg mL–1 ampicillin and 1 mM IPTG)
and incubated for 2 h at 30 °C. Oxidase activity was then assayed
following the protocol outlined previously.39 (link),53 (link) Briefly, the membrane containing colonies was transferred to a membrane
soaked in 0.1 mg mL–1 HRP (Sigma) and 100 mM potassium
phosphate pH 7.7 for 30 min (the prescreen). Colonies were then transferred
to a membrane soaked in 0.1 mg mL–1 HRP, DAB (Sigma),
2.5 mM α-methylbenzylamine (Sigma), and 100 mM potassium phosphate
pH 7.7. Oxidase activity was observed by the formation of a brown
DAB precipitate.
Colonies that exhibited the fastest color change
were picked and inoculated into LB (100 μg mL–1 ampicillin) and grown overnight (37 °C, 180 rpm), and the plasmids
were extracted using a plasmid miniprep kit (Qiagen). The sequencing
of variants was performed as above.
transformed into E. coli competent cells (T7 express,
New England Biolabs) and spread onto an LB agar with 100 μg
mL–1 ampicillin covered with a Hybond-N membrane
(Amersham Biosciences). Following incubation overnight at 30 °C,
the membrane containing single colonies was transferred to an LB agar
plate (100 μg mL–1 ampicillin and 1 mM IPTG)
and incubated for 2 h at 30 °C. Oxidase activity was then assayed
following the protocol outlined previously.39 (link),53 (link) Briefly, the membrane containing colonies was transferred to a membrane
soaked in 0.1 mg mL–1 HRP (Sigma) and 100 mM potassium
phosphate pH 7.7 for 30 min (the prescreen). Colonies were then transferred
to a membrane soaked in 0.1 mg mL–1 HRP, DAB (Sigma),
2.5 mM α-methylbenzylamine (Sigma), and 100 mM potassium phosphate
pH 7.7. Oxidase activity was observed by the formation of a brown
DAB precipitate.
Colonies that exhibited the fastest color change
were picked and inoculated into LB (100 μg mL–1 ampicillin) and grown overnight (37 °C, 180 rpm), and the plasmids
were extracted using a plasmid miniprep kit (Qiagen). The sequencing
of variants was performed as above.
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