To perform Northern blot, total RNA was extracted from leaves of 3-week-old dy1 mutant and WT plants. Ten microgram of total RNA was loaded for each lane and separated on 1.5% agarose gel. After separation, the RNA was blotted onto a positively charged nylon membrane (GE Healthcare, Pittsburgh, PA, United States). The probe for detecting DY1 transcripts was amplified using primer pair DY1-Probe-F and DY1-Probe-R, with DY1 cDNA as a template, and then labeled using DIG DNA Labeling Mix (Roche, Basel, Switzerland). Probing, washing, and detection of DY1 were performed according to the DIG High Prime DNA Labeling and Detection Starter Kit II user’s manual (Roche).
Dig dna labeling mix
Manufactured by Roche
Sourced in Switzerland
The DIG DNA Labeling Mix is a reagent used in molecular biology applications for the labeling of nucleic acids, such as DNA or RNA. It contains digoxigenin-labeled nucleotides that can be incorporated into synthesized DNA or RNA strands, allowing for their detection and analysis in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Dig easy hyb, by Roche (3 mentions)
Hybond n, by GE Healthcare (3 mentions)
Blocking one, by Nacalai Tesque (3 mentions)
Cdp star chemiluminescent substrate, by Roche (2 mentions)
Tripure reagent, by Aidlab (2 mentions)
Imagequant las 4000 system, by GE Healthcare (2 mentions)
Dig luminescent detection kit, by Roche (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Positively charged nylon membrane, by GE Healthcare (1 mentions)
Dig high prime dna labeling and detection starter kit 2, by Roche (1 mentions)
Nylon membrane, by Cytiva (1 mentions)
Dig easy hyb buffer, by Roche (1 mentions)
Cdp star, by Roche (1 mentions)
Las 3000 imaging system, by Fujifilm (1 mentions)
Alkaline phosphatase conjugated anti dig antibody, by Roche (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Nylon membrane, by GE Healthcare (1 mentions)
Dig luminescent detection kit for nucleic acids, by Roche (1 mentions)
Dig nucleic acid detection kit, by Roche (1 mentions)
Chemiluminescent nucleic acid detection module kit, by Thermo Fisher Scientific (1 mentions)
15 protocols using dig dna labeling mix
1
Detecting DY1 Transcripts via Northern Blot
2
Synthesizing Digoxigenin-Labeled Probes for Fgf3 and Fgf4
3
Genomic DNA Digestion and Southern Blot Analysis
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Genomic DNA (6 μg) purified from cultured cells was digested with BamHI and EcoRI, separated on 0.8% agarose gels and transferred to nylon membranes (Amersham). The membranes were incubated with digoxigenin (DIG)-labeled DNA probes in DIG Easy Hyb Buffer (Roche) at 42°C with constant agitation. After washing, an alkaline phosphatase-conjugated anti-DIG antibody (1∶10,000, Roche) was added to the membrane. Signals were raised by CDP-star (Roche) and detected by the LAS3000 imaging system (FUJIFILM). The Klf4 cDNA probe was generated using the DIG DNA labeling mix (Roche). The primers used in this experiment are listed in Table S7 .
4
Burkholderia Phage DNA Characterization
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Restriction profiles of the Burkholderia phages were analyzed by electrophoresis on 0.8% agarose gels. After separation of the DNA fragments, Southern blotting, and DNA-DNA hybridization were performed as previously described (Guerra et al., 2004 (link)). Digoxigenin-11-dUTP labeled phage DNA probes were prepared using the DIG DNA Labeling Mix (Roche Applied Science, Mannheim, Germany) according to the recommendations of the manufacturer.
5
Transgenic Porcine Genome Analysis
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Genomic DNA was extracted from skin samples of Tg fetuses using a DNA purification kit (DNeasy Blood and Tissue Kit; Qiagen). The purified genomic DNA (5 µg) was digested with PstI (Takara Bio, Shiga, Japan), separated by gel electrophoresis, and transferred onto a nylon membrane (Hybond N+; GE Healthcare Biosciences, Uppsala, UK). The membranes were blocked for 30 min at room temperature with blocking reagent (Blocking One; Nacalai Tesque, Kyoto, Japan). After blocking, the membranes were incubated in a hybridization solution (DIG Easy Hyb; Roche Diagnostics, Basel, Switzerland) and hybridized with a digoxigenin (DIG)-labeled Pdx1-Hes1 probe prepared by PCR using a DNA labeling reagent (DIG DNA Labeling Mix; Roche Diagnostics). The signals were developed and detected using conventional 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium chromogenic stain. The number of transgene copies integrated into the porcine genome was determined by comparison of the hybridization signal with that of the copy-number control, which was diluted to make a standard series (1–30 copies per diploid genome). For chromosomal analysis, fetal fibroblasts derived from a Tg pig with pancreatic agenesis were cultured and outsourced for fluorescence in situ hybridization (FISH) analysis (Chromosome Science Labo Inc, Sapporo, Japan).
6
RNA Extraction and Blotting Protocol
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For northern blotting and microarray analyses, we harvested cells and stored them at −80 °C until use. We extracted RNA as described previously [23 (link)], subjected it to electrophoresis, blotted it onto nylon membranes, and hybridised it with digoxigenin-labelled DNA probes [2 (link)] prepared using DIG DNA Labeling Mix (Roche, Mannheim, Germany) according to the manufacturer’s protocol.
7
Southern Blot for Xoo Strain Analysis
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The procedures of Southern blot were similar to a previously described method with some modifications46 (link). Briefly, 3.1-kb SphI fragment of plasmid pZWavrXa7 were gel purified after digestion. This fragment was labeled with DIG DNA Labeling Mix (Roche) or AP Direct Labeling and Detection kit (GE) as a probe. Twenty micrograms (for DIG-labeling probe) or 500 ng (for AP-labeling probe) of genomic DNAs from Xoo isolates were digested with SphI and used for hybridization. Size markers were visualized by adding DIG/AP labels. Three independent repeats were performed in each sample and the phylogenetic tree was generated as described in RAPD analyses.
8
Transcriptome Analysis of Fish Samples
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Total RNA was extracted from fish samples using TRIpure Reagent (Aidlab, RN0102) according to the manufacturer’s instructions. Digoxigenin (DIG)-labeled 5′ETS-1, ITS1, and ITS2 probes were obtained by PCR with specific primers (S1 Table ) and the corresponding plasmid DNA as the template, together with the DIG DNA Labeling Mix (Roche Diagnostics,11277065910). Northern blot hybridization was performed as previously described [18 (link)]. DIG-labeled probes were detected with CDP-Star Chemiluminescent Substrate (Roche, Cat#12041677001), according to the manufacturer’s instructions.
9
Screening Targeted ES Cells for R26-Sall4 Mice
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To screen targeted ES cells, a DNA fragment described in [33 (link)] was labelled using DIG DNA labeling mix (Roche, Cat#11277065910) and genomic Southern hybridization was performed following a standard procedure [35 ]. Genomic PCR of ES cell DNA and PCR genotyping on tail clip DNA for the R26-Sall4 mice were conducted as previously described [33 (link)].
10
Detecting Transgene Copy Numbers by Southern Blot
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Transgene copy numbers were detected by Southern blot hybridization analysis. Approximately 12 μg of total DNA was digested and blotted onto nylon membranes (GE Healthcare, UK). A 477-bp LRP gene fragment was amplified using 5′-ATGGGTGTTTTCACATATG-3′ and 5′-TCAATTGTATTCAGGATGG-3′ to generate the hybridization probe by DIG DNA Labeling Mix (Roche, Switzerland). Detection was performed using a DIG Luminescent Detection Kit for Nucleic Acids (Roche, Switzerland). The detailed procedure was according to the manual.
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