PCR amplification with primers targeting Rs-fliC gene was performed according to Schönfeld et al. (2003) (link) from total community DNA from rhizosphere and shoots of tomato plants grown in B3B-infested soils. PCR products were analyzed by 1% agarose gel electrophoresis for 1 h (50 V), gels were checked by UV light after staining with ethidium bromide and Southern-blotted as described by Binh et al. (2008) (link). Hybridization was performed with Digoxygenin-labeled fliC probe generated from purified PCR products obtained with B3B by means of the DIG DNA labeling kit (Roche Applied Science, Mannheim, Germany).
Dig dna labeling kit
Manufactured by Roche
Sourced in Germany, Switzerland
The DIG DNA labeling kit is a laboratory tool used for the incorporation of digoxigenin (DIG) labels into DNA samples. The kit provides the necessary reagents and protocols to enable the sensitive detection and analysis of DNA molecules through various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta confocal microscope, by Zeiss (4 mentions)
Cdp star, by Roche (3 mentions)
Amersham hybond n membrane, by GE Healthcare (3 mentions)
Dnase, by Thermo Fisher Scientific (3 mentions)
Dig nucleic acid detection kit, by Roche (2 mentions)
Nylon membrane, by Roche (2 mentions)
Dig luminescent detection kit, by Roche (2 mentions)
Cpd star, by Roche (2 mentions)
Wizard genomic dna purification kit, by Promega (2 mentions)
Hybond n nylon membrane, by GE Healthcare (2 mentions)
Ecori, by Thermo Fisher Scientific (2 mentions)
Anti gfp, by Abcam (1 mentions)
Nicotinamide adenine dinucleotide, by Merck Group (1 mentions)
Poly d lysine pdl, by Merck Group (1 mentions)
Cytotoxic detection kit, by Roche (1 mentions)
2 3 bis 2 methoxy 4 nitro 5 sulfophenyl 2h tetrazolium 5 carboxanilide inner salt xtt, by Merck Group (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Polymyxin b, by Merck Group (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
37 protocols using dig dna labeling kit
1
PCR-based Detection of Rs-fliC Gene
2
Protein and DNA Localization in Drosophila Ovaries
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The combination of protein and DNA localization was performed according to a previously described procedure (60 (link)). The HeT-A probe used for DNA FISH was a cloned fragment of the HeT-A ORF (61 (link)). The probe corresponding to 2R-3R TAS was a cloned PCR fragment obtained using the primers listed in Supplementary Table S4 . The probes were labeled using a DIG DNA labeling kit (Roche). Rat anti-Rhi antibodies (58 (link)) were used. To stain the DNA, the ovaries were incubated in PBS containing 0.5 μg/ml 4',6-diamidino-2-phenylindole (DAPI). Two biological replicates were obtained for each experiment. The Zeiss LSM 510 Meta confocal microscope was used for visualization. Confocal image z-stacks generated with the slice step of 1.5 μM were used for the statistics.
Immunostaining was carried out according to the previously described procedure (62 (link)). The following primary antibodies were used for immunostaining: mouse anti-Not1 (6 (link)) (kindly provided by E. Wahle), rat anti-HOAP (polyclonal antibodies against full-length HOAP protein were generated by the Immunochemistry laboratory, Branch of IBC RAS, Pushchino, Russia), guinea pig anti-HipHop (kindly provided by Y. Rong), rabbit anti-Ccr4 (6 (link)) (kindly provided by M. Simonelig), mouse anti-Piwi (kindly provided by J. Brennecke), anti-GFP (Abcam).
Immunostaining was carried out according to the previously described procedure (62 (link)). The following primary antibodies were used for immunostaining: mouse anti-Not1 (6 (link)) (kindly provided by E. Wahle), rat anti-HOAP (polyclonal antibodies against full-length HOAP protein were generated by the Immunochemistry laboratory, Branch of IBC RAS, Pushchino, Russia), guinea pig anti-HipHop (kindly provided by Y. Rong), rabbit anti-Ccr4 (6 (link)) (kindly provided by M. Simonelig), mouse anti-Piwi (kindly provided by J. Brennecke), anti-GFP (Abcam).
3
Comprehensive Cytotoxicity Evaluation Protocol
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Restriction enzymes were purchased from New England Biolabs (USA). Anti-digoxigenin (DIG) Fab fragment conjugated with horseradish peroxidase (HRP), a DIG DNA labeling kit, and cytotoxic detection kit (LDH) were obtained from Roche (Switzerland). Hoechst 33258, nicotinamide adenine dinucleotide (NAD), poly-D-lysine (PDL), polymyxin B, and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) were obtained from Sigma-Aldrich (USA). Bradford reagent and γ-globulin standard were purchased from Bio-Rad Laboratories (USA). A limulus amebocyte lysate (LAL) assay kit was obtained from Cambrex Bio Science (USA). Medium, antibiotics and supplementary components for cell culture were purchased from Gibco (USA). Bacterial culture materials were purchased from Becton Dickinson (USA).
4
Northern Blot Analysis of SAMD12-AS1 in HepG2 Cells
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Total RNA from HepG2 and HepG2-4D14 cells was extracted using TRIzol reagent. Northern blot was performed according to the manufacturer’s instructions for the NorthernMax-Gly Kit (Invitrogen, USA) as previously described20 (link). The probe for SAMD12-AS1 (nt 200–500) was labeled with digoxigenin using the DIG DNA Labeling Kit (Roche, Switzerland), and chemiluminescent detection was performed using the DIG Chemiluminescent Detection Kit (Roche).
5
HPV DNA Detection in Tissue Sections
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Slides of formalin fixed, paraffin embedded raft tissue were baked and de-paraffinised by washing three times in xylene followed by two washes in 100% ethanol. The slides were then rehydrated in 80%, 50% and 30% ethanol for two minutes each, and then finally in PBS for 5 minutes. Sections were then heated in 0.01M citric acid (pH 6.0) in microwave for 2 minutes, prior to protein digestion with Digest All 3 pepsin (Zymed) at 37°C for 10 seconds. HPV16 or 18 DNA was labelled with digoxigenin by use of the DIG DNA labeling Kit (Roche Applied Science Gmbh, Germany) according to the manufacturer’s instructions. DIG-Labeled HPV DNA was added to slides, denatured for 5 min at 76°C, and incubated overnight at 42°C. After washing, the DIG-positive HPV probe was detected with an anti-digoxigenin-POD antibody at 1:400 (Roche Applied Science Gmbh, Germany) and the signal amplified using a Tyramide Signal Amplification Kit (Perkin-Elmer Ltd, UK) according to the manufacturer's instructions. E1^E4 protein and nuclei were identified with anti-16E1^E4 antibody TVG 405 conjugated to Alexa 488 and DAPI (4', 6-diamidino-2-phenylindole).
6
DNA Digestion and Southern Blot Analysis
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One hundred microgram of genomic DNA was digested overnight at 37°C with XhoI or with BglII and SalI. Digested DNA was separated on 0.7% agarose gels by electrophoresis (50 V, 60 min) and transferred to Hybond-N+ nylon membranes (GE Healthcare). Probes A and B were synthesized using a DIG DNA Labeling Kit (Roche Applied Science) according to the manufacturer’s instructions, with primers 7 and 8 (probe A), primers 9 and 10 (probe B) in S1 Table . Each membrane was incubated at a different temperature depending on the combination of probe and enzymes for DNA digestion; at 45°C for probe A and XhoI, and 42°C for probe B with BglII and SalI. Then, all signals were detected using a DIG Nucleic Acid Detection Kit (Roche Applied Science).
7
Cloning and Sequencing Mbol-Digested DNA
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PSU genomic DNA was digested with the restriction endonuclease (RE) MboI, according to the manufacturers’ instructions (Invitrogen Life Technologies), resulting in a smear with DNA fragments ranging between 3 kb and 100 bp. The restriction products were later cloned using routine procedures (FERMENTAS Life Science, Invitrogen Life Technologies). A part of the obtained colonies were transferred onto a nylon membrane Hybond-N+ (Amersham, GE Healthcare) and the DNA on the membrane probed to MboI restriction products labeled with digoxigenin-11-dUTP, using DIG DNA labeling Kit (Roche Diagnostics). Hybridization was performed at 68 °C. The positive signals were visualized using the chemiluminiscent CDP-Star system (Roche Diagnostics). Plasmidic DNA of the positive clones was isolated using the High Pure Plasmid Isolation kit (Roche Diagnostics) and sequenced in both directions using M13 primers.
8
Molecular Genetic Analysis of Lentinula edodes
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A cDNA probe was labeled with digoxigenin (DIG) by the random hexamer procedure using a DIG DNA labeling kit (Roche Diagnostics, Mannheim, Germany). Genomic DNA was extracted from L. edodes mycelia with 2.0% Cetyl trimethyl ammonium bromide (CTAB) in 100 mM Tris–HCl, pH 8.0, 1.4 M NaCl, 20 mM EDTA, and 0.2% β-mercaptoethanol, and total RNA was extracted with ISOGEN (Nippon Gene, Toyama, Japan) from L. edodes mycelia or transformants with the RNAi vector for Le-Dd10. Southern blotting and Northern blotting analyses were conducted using each cDNA as a probe. Twenty μg of genomic DNA digested with an appropriate restriction enzyme and ten μg of total RNA were separated on an agarose gel. After agarose gel electrophoresis, agarose gels were transferred onto Hybond™ N+nylon membrane (GM Healthcare) with VacuGeneXL (GM Healthcare). Hybridization was carried out at 42 °C for Southern blotting analysis and at 50 °C for Northern blotting analysis in DIG easy hyb (Roche Diagnostics).
9
Tissue-Specific Localization of HSP40 mRNA
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The cellular localization of HSP40 mRNA were performed in selected tissues with high transcript levels by in situ hybridization (ISH). Briefly, samples from the muscle, gill, hepatopancreas and ovary were removed quickly and fixed overnight in 4% paraformaldehyde in PBS. Samples were dehydrated in an ethanol series, immersed in xylene, embedded in paraffin, cut into 5 μm sections and mounted onto slides. A digoxin (DIG)-labeled DNA probe of 585 bp (Supplementary Data 2 ) against Lv-HSP40 mRNA was synthesized with the DIG DNA Labeling Kit (Roche). The sections were deparaffinized, prehybridized and hybridized with the DIG-labeled DNA probe (0.4 ng/ml) at 42°C overnight as described previously (Chen et al., 2008 (link)). After that, the sections were incubated with horseradish peroxidase (HRP)-conjugated anti-DIG antibody (Roche), and the ISH signal were developed by a diaminobenzidine (DAB, MXB Biotechnologies) reaction and observed with a Leica DM-IRB light microscopy (Leica).
10
Genomic DNA Hybridization Assay
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To perform DNA hybridization, 5 μg genomic DNA for each sample was digested with EcoR I (New England, Ipswich, MA) at 37 °C for 8 h. The digested DNAs were separated by electrophoresis on a 0.8% (w/v) agarose gel at 45 v for overnight and transferred onto a Hybond N+ membrane (GE Healthcare, Little Chalfont, UK). Primers targeting the reverse transcriptase region of the RTEs (supplementary table S5 , Supplementary Material online) were used to amplify the fragments. About 500-ng purified PCR products were used as probes and labelled with the DIG DNA labeling kit (Roche, Mannheim, Germany). Hybridization was performed using the “DIG easy hyb” system by following the manufactures instructions.
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