A total of 1 µg DNA was digested with MluI-HF restriction endonuclease (New England Biolabs, Frankfurt am Main, Germany) for cleavage of nuclear DNA. This restriction endonuclease does not have a cutting site on the mitochondrial genome. DNA was separated, along with DIG-labeled DNA Molecular Weight Marker II (Roche, Mannheim, Germany), in a 0.6% agarose gel containing 1.25 µM ethidium bromide at 40 V overnight. The gels were alkaline treated and neutralized before the DNA was blotted to Zeta-Probe membranes (Bio-Rad, Hercules, CA, USA) and immobilized by baking at 80 °C for 30 min. Blots were hybridized overnight at 48 °C with PCR-generated digoxigenin-labeled probes. Probes were synthesized using the PCR DIG Probe Synthesis Kit (Roche, Mannheim, Germany) with primers 5′-TCATCCCTGTAGCATTGTTCG-3′ and 5′-GAAGAACTGATTAATGTTTGGGTCT-3′ for the MT-ND5 gene in the region 12602–12690 in the mitochondrial genome, or primers 5′-GTTGGTGGAGCGATTTGTCT-3′ and 5′-GGCCTCACTAAACCATCCAA-3′ for nuclear 18S rRNA genes. Chemiluminescent detection with anti-DIG-AP antibody Fab fragment (Roche, Mannheim, Germany) and CSPD (Roche, Mannheim, Germany) was performed, and the signal was recorded on a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA) [12 (link)].
Zeta probe membrane
Manufactured by Bio-Rad
Sourced in United States
The Zeta-Probe membrane is a specialized filtration membrane used in various laboratory applications. It is designed to provide efficient and reliable separation of molecules, particles, or other analytes from complex mixtures. The core function of the Zeta-Probe membrane is to facilitate high-performance filtration and sample preparation, enabling effective isolation and purification of the desired components.
Automatically generated - may contain errors
Lab products found in correlation
Expresshyb hybridization buffer, by Takara Bio (6 mentions)
Ultrahyb, by Thermo Fisher Scientific (5 mentions)
Tri reagent, by Merck Group (4 mentions)
Cl xposure film, by Thermo Fisher Scientific (4 mentions)
Amersham gene images alkphos direct labeling and detection system, by GE Healthcare (4 mentions)
Pcr dig probe synthesis kit, by Roche (3 mentions)
Typhoon fla 9000, by GE Healthcare (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Imagequant, by GE Healthcare (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Anti dig ap antibody fab fragment, by Roche (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
α 32p dctp, by PerkinElmer (2 mentions)
T4 polynucleotide kinase, by New England Biolabs (2 mentions)
Maxiscript t7 kit, by Thermo Fisher Scientific (2 mentions)
Ribopure kit, by Thermo Fisher Scientific (2 mentions)
Storm scanner, by GE Healthcare (2 mentions)
Iqtl 8.0 quantification software, by GE Healthcare (2 mentions)
α 32p datp radprime dna labeled, by Thermo Fisher Scientific (2 mentions)
Trans blot sd semidry transfer apparatus, by Bio-Rad (2 mentions)
80 protocols using zeta probe membrane
1
Mitochondrial DNA Digestion and Analysis
2
XBP-1 Transcriptional Activity Assay
3
RNA Extraction and Biotinylation Protocol
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RNA was extracted from the plant samples using the RNeasy Plant (QIAGEN Germantown, MD, USA). The extracted labeled RNA was then biotinylated and separated on streptavidin columns as previously described [8 (link)]. The separated pre-existing RNA was tested by dotting onto Zeta probe membranes (Biorad Hercules, CA, USA) and detecting any remaining biotinylated RNA using streptavidin-HRP (Genscript), ECL plus reagents (GE Life Sciences, Little Chalfont, Buckinghamshire, UK) and a light detection system. RNA samples were checked for integrity using Agilent Bioanalyzer pico or nanochips, according to the manufacturer’s protocols.
4
Molecular Analysis of papR and C271
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Total RNA was isolated using heavy phase lock gel tubes (5Prime) and Northern blot analysis of papR (JMJ834), C271 (JMJ767) and 5S was performed as described previously [88 (link)]. Samples were electrophoresed along with a RiboRuler low range RNA ladder (Thermo Scientific) and transferred onto Zeta-probe membranes (Bio-Rad). Membranes probed with radiolabelled primers were visualized using a Typhoon FLA9500 scanner (GE Healthcare) and bands quantified where appropriate using Image Studio Lite Version 4.0 software. Primer extension analysis was carried out as described by Franch et al.[89 (link)] using primers JMJ832+JMJ833 to amplify PapR with upstream and downstream flanking regions by PCR to serve as template for the sequencing reaction and radiolabelled JMJ834 was used in both sequencing and reverse transcription reactions. Relevant primers are listed in S3 Table .
5
Dot-Blot RNA Quantification Assay
6
Southern Blot of Mitochondrial and Nuclear DNA
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One microgram of total DNA was digested with BamHI restriction endonuclease (Fermentas). Products were separated on a 0.6% agarose gel at 40 V overnight along with DIG-labeled DNA Molecular Weight Marker II (Roche). The gels were alkaline treated and neutralized. DNA was blotted to Zeta-Probe membranes (Bio-Rad) and immobilized by baking at 80 °C for 30 min. Blots were hybridized with PCR-generated digoxigenin-labeled mitochondrial and nuclear (18S rRNA) probes (PCR DIG Probe Synthesis Kit, Roche; Supplementary Table 4 ) overnight at 48 °C. Chemiluminescent detection with anti-DIG-AP antibody Fab fragment (Roche) and CSPD (Roche) was performed and signal was recorded on a ChemiDoc Imaging System (Bio-Rad). If required, blots were stripped with 0.2 M NaOH + 0.1% SDS at 37 °C. Uncropped scans of the most important blots are shown in Supplementary Fig. 8 .
7
In vitro RNA Binding Assay
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T7 in vitro transcription (NEB) was performed for eutR and mavR incorporating Bio-11-UTP (Fisher) where indicated. Transcripts were purified using NucAway Spin Columns (Invitrogen). Purified transcripts were incubated at the indicated concentrations in 1× structure buffer (Ambion RNase T1 kit), 2 ng/µl yeast RNA (Ambion) and brought to a total volume of 20 μl with RNase-free water. Samples were incubated at 85°C for 3 min followed by 20 min at 37°C. Following the addition of 5× RNA loading dye (50% glycerol, 0.1% bromophenol blue), samples were subjected to electrophoresis on 5% native TBE gels using 1× TBE as running buffer. Electrophoresed samples were transferred by capillary action to Zeta-probe membranes (BioRad) in 20× SSC. After UV crosslinking the RNA to the membrane, the membranes were subjected to the Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Scientific). EMSAs were visualized using a Gel Doc XR + Gel Documentation System.
8
Analyzing Mouse Mitochondrial DNA
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mtNA was treated with the restriction enzyme BlpI, which cleaves mouse mtDNA once, followed by treatment with Proteinase K, extraction with phenol and CI and alcohol precipitation. Samples were then incubated with 10 units of McrBC (New England Biolabs) in the buffer provided by the company in the presence or absence of 1 mM GTP in 20 µl reaction volumes at 37 °C for 4 h. Reaction mixtures were then treated with Proteinase K and were electrophoresed in agarose gels, and the gels were processed for Southern hybridisation and blotted onto Zeta-Probe Membranes (Bio-Rad Laboratories). After fixation of blotted DNA using UV cross-linking, Southern hybridisation was performed using radiolabelled probes as described previously45 (link) with minor modifications. Membranes were then exposed to phosphorimaging plates and bands were visualised and quantified using a Typhoon FLA9500 instrument (GE Healthcare). DNA probes were radiolabeled using Random Primer DNA Labeling Kit Ver.2 (TaKaRa) and [α-32P] dCTP (3000 Ci/mmol) (PerkinElmer) using PCR fragments of mouse mtDNA spanning nt 16,033–15,510.
9
Genomic DNA Extraction and Analysis
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Genomic DNA was extracted using glass acid beads (Sigma), phenol: cholorform: isoamyl alcohol (25:24:1) (Sigma) and RNAse A treated (Fisher). Following centrifugation, pellets were precipitated with 0.05 mM Sodium Acetate (Sigma) and Ethanol (Fisher) at −20 °C during 30 minutes and resuspended in water.Genomic DNA was then digested with corresponding enzymes and run in 1% agarose gel. DNA was transferred to a nylon membrane (Zeta probe membranes, Bio-Rad), probed with DIG probes (Roche) and hybridized as described57 (link).
10
Northern Blotting of tRNA Isoacceptors
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For deacylation immunoprecipitated samples were incubated with 125 mM Tris–HCl, pH 9.0, 0.1 M EDTA, 0.5% (w/v) SDS at room temperature for 45 min, before neutralization with an equal volume of 1 M NaOAc, pH 5.5. RNA was extracted twice with 5:1 acidic phenol:chloroform, precipitated with ethanol, and resuspended in ddH2O. Samples containing 150 ng of total RNA were separated on 8M urea-containing 8% polyacrylamide gels followed by electroblotting to Zeta-probe membranes (Bio-Rad). The blots were sequentially probed for B. subtilis tRNAAla, B. subtilis tRNALys and E. coli tRNAVal using 32P-labeled oligonucleotides (Supplementary Table S2 ). The probes for B. subtilis tRNAAla were designed to hybridize with the conserved TΨC-loop of both tRNAAla isoacceptors. Signals were detected by phosphorimaging using a Typhoon FLA 9500 biomolecular imager. In parallel, a second gel was subjected to SYBR Gold (Life Technologies) nucleic acid staining for 30 min, followed by visualization using a Typhoon Trio Variable Mode Imager (Amersham Biosciences).
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