Genomic DNA (~2.5 to 5 g) was digested with Bgl II and Pst I, electrophoresed on an 0.8% agarose gel at 25 V for ~18 hours, blotted onto a BrightStar-Plus Positively Charged Nylon Membrane (Thermo Fisher Scientific) in 0.4 M NaOH and 0.6 M NaCl transfer solution, and then baked at 80°C for 2 hours. The membranes were hybridized overnight at 42°C using an Eg fp fragment labeled by DIG DNA Labeling and Detection Kit (Roche) as the probe. The membranes were washed at 42°C in 0.1× saline sodium phosphate EDTA and 0.1% sodium dodecyl sulfate (Roche) until the background signal was eliminated. The signal was detected with the DIG DNA Labeling and Detection Kit (Roche), and images were captured using a Bio-Rad ChemiDoc XRS+ System.
Dig dna labeling and detection kit
Manufactured by Roche
Sourced in Germany, United States, Switzerland, France, China, Canada
The DIG DNA Labeling and Detection Kit is a product designed for the labeling and detection of DNA. The kit provides the necessary reagents and protocols to incorporate a digoxigenin (DIG) label into DNA samples, which can then be detected using anti-DIG antibodies. The core function of this kit is to enable the labeling and subsequent identification of DNA molecules.
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Lab products found in correlation
Pcr dig probe synthesis kit, by Roche (12 mentions)
Positively charged nylon membrane, by Roche (9 mentions)
Hybond n membrane, by Cytiva (5 mentions)
Nylon membrane, by Roche (5 mentions)
Hybond n, by Cytiva (4 mentions)
Hybond n nylon membrane, by GE Healthcare (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Ecori, by New England Biolabs (2 mentions)
Versadoc imaging system, by Bio-Rad (2 mentions)
Dig luminescent detection kit for nucleic acids, by Roche (2 mentions)
Dig easy hyb, by Roche (2 mentions)
Hybond n, by Merck Group (2 mentions)
Stratalinker, by Merck Group (2 mentions)
Immobilon ny membrane, by Merck Group (2 mentions)
Nylon membrane, by Bio-Rad (2 mentions)
Glucanex, by Novozymes (2 mentions)
Tianamp bacteria dna kit, by Tiangen Biotech (2 mentions)
Zeta probe, by Bio-Rad (2 mentions)
Plant genomic dna kit, by Tiangen Biotech (2 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (2 mentions)
104 protocols using dig dna labeling and detection kit
1
Genomic Southern Blot Analysis
2
Detecting ARS Plasmid Integration via Southern Blot
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To detect potential integration events of the ARS plasmid, Southern blot was performed. 20 µg of the gDNA preparations was also used for qPCR and an additional preparation from the empty background strain K. phaffii BSY11G1, as well as 10 ng and 200 ng of isolated plasmid were loaded on an 0,75% agarose gel without ethidium bromide (80 V for 2 h). A second gel was prepared with Ethidium bromide and 0.5 µg of the gDNA samples as well as 10 ng and 200 ng of isolated plasmid were loaded on the gel. For detection of the reporter gene CalB, a probe was produced by PCR with primers 28 & 32 and the plasmid as a template and was DIG-labeled using the DIG DNA Labeling and Detection Kit (Roche, Mannheim, Germany) following the manufacturer’s manual. The blotting and hybridization steps were performed as described by Green and Sambrook [59 (link), 60 (link)] in an aqueous hybridization buffer and with a DIG-labeled probe. The probe was detected using the DIG DNA Labeling and Detection Kit.
3
Gene Disruption and Complementation Protocol
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Gene disruption and complementation were conducted as previously described (9 (link)). Plasmids p0380-bar and p0380-sur-gateway were used to construct gene disruption and complementation vectors. First, the 5′ and 3′ flanking fragments of genes were amplified from the WT genomic DNA with paired primers P1/P2 and P3/P4 and inserted into the BamHI-EcoRI and XbaI-SpeI sites of p0380-bar, respectively. The resultant plasmid was used to disrupt the target gene. To prepare the complementation vector, the full-length gene with promoter sequence was cloned from the WT genomic DNA with primers P7 and P8. The resulting fragment was cloned into the vector p0380-sur-gateway. The method of Agrobacterium-mediated transformation was used to transform the disruption vector into the WT strain and complementary vector into the gene disruption mutant. Putative deletion and complementation mutants were screened by phosphinothricin (200 μg/mL) and chlorimuron ethyl (10 μg/mL), respectively. Candidate transformants were prescreened by PCR with the primer pair P5/P6. To further confirm, Southern blotting was performed with a digoxigenin (DIG) DNA labeling and detection kit (Roche, Germany). A fragment amplified with the primers P9 and P10 was used to prepare the probes.
4
Intron Insertion Detection in C. perfringens
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DNA was extracted from all C. perfringens strains using the MasterPure Gram-positive DNA purification kit. PCR for the virS or agrB genes was performed using the primers described in the previous section. For the wild-type strains, the sizes of PCR products are listed in Table 4 . For the null mutant strains, PCR using the same pair of the primers should amplify a product from the intron-disrupted gene that is ∼900 bp larger than the gene from the wild-type strains. All PCR conditions were described previously (25 (link), 30 (link)).
For Southern blot analysis to detect an intron insertion, aliquots (3 μg each) of wild-type, single-null, or double-null mutant strain DNA were digested overnight with EcoRI at 37°C according to the manufacturer’s instructions (New England Biolabs). The digested DNA samples were then electrophoresed on a 1% agarose gel before transfer onto a positively charged nylon membrane (Roche) for hybridization with an intron-specific probe (20 (link)). The intron-specific probe was prepared using the PCR digoxigenin (DIG) probe synthesis kit (Roche) and intron primers (IBS and EBS2). After hybridization, Southern blots were developed using reagents from the DIG DNA labeling and detection kit (Roche) according to the manufacturer’s instructions.
For Southern blot analysis to detect an intron insertion, aliquots (3 μg each) of wild-type, single-null, or double-null mutant strain DNA were digested overnight with EcoRI at 37°C according to the manufacturer’s instructions (New England Biolabs). The digested DNA samples were then electrophoresed on a 1% agarose gel before transfer onto a positively charged nylon membrane (Roche) for hybridization with an intron-specific probe (20 (link)). The intron-specific probe was prepared using the PCR digoxigenin (DIG) probe synthesis kit (Roche) and intron primers (IBS and EBS2). After hybridization, Southern blots were developed using reagents from the DIG DNA labeling and detection kit (Roche) according to the manufacturer’s instructions.
5
Isolation and Identification of crAss-like Phages
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CrAss1-ORF46 probe is a digoxigenin (DIG)-labeled 50-bp probe (5’-ACCTGCTTCTACACTTTCCTTAGATGAACTAATATCTAACCCAGCTCTAT-3’) located in the ΦCrAss001 genome and commercially available. Plaques observed in the soft agar layer were transferred to a nylon membrane (Hybond N + , Amersham Pharmacia Biotech) and hybridized with the probe. Hybridization was performed at 53 °C, according to the standard procedure46 . Stringent hybridization was achieved with the DIG-DNA Labeling and Detection Kit (Roche Diagnostics) according to the manufacturer’s instructions.
Plaques showing a positive signal were recovered from the soft agar overlayer with a sterile loop, resuspended in 200 µl of SM buffer and submitted to a chloroform treatment to eliminate bacterial cells. The isolated plaques confirmed to be crAss-like phages by qPCR were further propagated in larger volumes of B. intestinalis culture for subsequent analysis.
Plaques showing a positive signal were recovered from the soft agar overlayer with a sterile loop, resuspended in 200 µl of SM buffer and submitted to a chloroform treatment to eliminate bacterial cells. The isolated plaques confirmed to be crAss-like phages by qPCR were further propagated in larger volumes of B. intestinalis culture for subsequent analysis.
6
Isolation and Identification of crAss-like Phages
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CrAss1-ORF46 probe is a digoxigenin (DIG)-labeled 50-bp probe (5’-ACCTGCTTCTACACTTTCCTTAGATGAACTAATATCTAACCCAGCTCTAT-3’) located in the ΦCrAss001 genome and commercially available. Plaques observed in the soft agar layer were transferred to a nylon membrane (Hybond N + , Amersham Pharmacia Biotech) and hybridized with the probe. Hybridization was performed at 53 °C, according to the standard procedure46 . Stringent hybridization was achieved with the DIG-DNA Labeling and Detection Kit (Roche Diagnostics) according to the manufacturer’s instructions.
Plaques showing a positive signal were recovered from the soft agar overlayer with a sterile loop, resuspended in 200 µl of SM buffer and submitted to a chloroform treatment to eliminate bacterial cells. The isolated plaques confirmed to be crAss-like phages by qPCR were further propagated in larger volumes of B. intestinalis culture for subsequent analysis.
Plaques showing a positive signal were recovered from the soft agar overlayer with a sterile loop, resuspended in 200 µl of SM buffer and submitted to a chloroform treatment to eliminate bacterial cells. The isolated plaques confirmed to be crAss-like phages by qPCR were further propagated in larger volumes of B. intestinalis culture for subsequent analysis.
7
Transgene Copy Number Detection
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The copy number of the T1 transgenic lines with positive results was identified by Southern blot. First, genomic DNA was extracted and digested with the restriction enzyme EcoR Ι. Then, the digested DNA was fractionated on a 0.8 % agarose gel and transferred to nylon membranes. The PCR product of NPTII was used as the probe. Standard procedures for Southern blot analysis, probe labeling, and detection were used following the DIG DNA Labeling and Detection Kit (Roche).
8
Genetic Manipulation Techniques in Vibrio
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Plasmid isolation, restriction digestion, DNA ligation, transformation, DNA extraction, and PCR were carried out using standard protocols, as described elsewhere (Green and Sambrook, 2012 ). Restriction enzymes, T4 DNA ligase, DNA polymerase, and the DIG-DNA labeling and detection kit were from Roche (Roche NimbleGen, RRID:SCR_008571 ). The oligonucleotides used for this work are listed in Supplementary Material 1 and were purchased from Invitrogen (Molecular Probes, RRID:SCR_013318 ). Mutants of the V. spinosum recA promoter (VSP_RS32310) were obtained using oligonucleotides carrying designed substitutions (Supplementary Material 1 ). The DNA sequence of generated fragments was verified by sequencing (Macrogen, RRID:SCR_014454 ).
9
AAV8 Genome Integration Assessment
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To test whether AAV8 integrated into the genome of offspring, we applied a previously reported method.10 (link) Total DNA was extracted from the tail using a TIANamp Genomic DNA kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. Genome integrations were quantified by PCR using specific primers for the CAG promoter and the inserted Lhcgr. PCR was performed using a Bio-Rad T100 following the manufacturer’s protocol. The primers used for PCR are listed in Table S2 . To detect viral DNA by Southern blotting, we digested 10 μg of DNA from offspring and AAV-Lhcgr plasmid with the indicated restriction enzymes and separated the fragments by electrophoresis on a 0.7% agarose gel. DNA was transferred and blotted onto a Hybond N+ nylon membrane (Amersham Biosciences, UK). The probe targeting AAV was synthesized and labeled with digoxigenin-dUTP (PCR DIG Probe Synthesis Kit, Roche). Hybridization was performed by Zoonbio Biotechnology Co., Ltd (Nanjing, China) using a DIG DNA labeling and detection kit (Roche). The hybridized membrane was detected in the presence of CSPD (C18H20ClNa2O7P), and the hybridized signals were visualized on X-ray film. The primers used for probe synthesis are listed in Table S2 .
10
Northern Blot Analysis of Ccdc181 Expression
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