Southern blotting was performed as described previously [23 (link)]. Briefly, 30 μg of genomic DNA was digested with EcoRV and StuI at 37°C and electrophoresed on a 0.8% agarose gel at 15 V for 14–18 h. Subsequently, DNA fragments were transferred to an Immobilon-Ny+ Transfer Membrane (Millipore, Concord Road, Billerica, MA, USA) by an alkaline transfer technique, followed by hybridization for >18 h at 55°C in hybridization buffer. The probe used was PCR amplified with primers HPRT 3′probe Fw and HPRT 3′probe Rv (Additional file 1 : Table S1). Southern hybridization was performed using Amersham Gene Images AlkPhos Direct Labelling and Detection System (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). Signals were detected with CDP-Star (Roche, Basel, Switzerland), and analyzed by using the Fuji Image Analyzer LAS-1000UVmini (Fuji Film Co., Tokyo, Japan).
Amersham gene images alkphos direct labelling and detection system
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The Amersham Gene Images AlkPhos Direct Labelling and Detection System is a lab equipment product that enables the direct labelling and detection of nucleic acid samples. It provides a method for the non-radioactive detection of DNA and RNA.
Automatically generated - may contain errors
Lab products found in correlation
Hybond n nylon membrane, by Cytiva (2 mentions)
Cdp star chemiflourescent detection system, by GE Healthcare (2 mentions)
Immobilon ny transfer membrane, by Merck Group (1 mentions)
Fuji image analyzer las 1000uvmini, by Fujifilm (1 mentions)
Cdp star, by Roche (1 mentions)
Chef dr 2 system, by Bio-Rad (1 mentions)
Genescreen plus hybridization transfer membrane, by PerkinElmer (1 mentions)
5 protocols using amersham gene images alkphos direct labelling and detection system
1
Genomic DNA Southern Blotting Protocol
2
Mitochondrial DNA Verification in Kudoa Infection
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To verify that the obtained genome sequence was derived from the mitochondrial chromosome and not from the nuclear copies of mitochondrial DNA (in nuclear chromosomes) or from fish mitochondria, we performed Southern blotting. We performed pulsed field electrophoresis of K. septempunctata isolate 0904 total DNA and Kudoa-free Paralichthys olivaceus total DNA (control) on a 1% agarose gel in 0.5x TBE. Using CHEF-DR II system (Bio-Rad), the electrophoresis was performed under 6 V/cm, 120° angle in two blocks: the run time was 3 h with 0.1–1 sec switch time ramp, followed by 11 h with 1–2 sec switch time ramp. DNA was transferred from the gel to a GeneScreen Plus Hybridization Transfer Membrane (PerkinElmer) by the alkaline transfer method. DNA probes were labeled using Amersham Gene Images AlkPhos Direct Labelling and Detection System (GE Healthcare) and hybridized to the DNA on the membrane. The probes were PCR products amplifying the region at 10194–11194 bp (within cox1 gene) or the region at 17170–17760 bp (no gene) of K. septempunctata isolate 0904 genome.
3
Characterization of Vea1 gene in Aspergillus
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A 1052-bp probe to the 5’ UTR of VEA1 was amplified from G217B ura5− genomic DNA using primer set OAS6659/OAS6660. A second 1013-bp probe to the VEA1 CDS was amplified from G217B ura5− genomic DNA using primer set OAS6663/OAS1969. In separate reactions the resulting PCR fragments were coupled with an alkaline phosphatase enzyme using the Amersham Gene Images AlkPhos Direct Labelling and Detection System (GE Healthcare), according to the manufacturers protocol. Genomic DNA from the parental strain G217B ura5− and the mutant strains was isolated by phenol chloroform extraction. In duplicate, 15 µg of genomic DNA from both samples was digested with restriction enzyme BglII for 1 hour at 37˚C. Digested DNA was size separated by gel electrophoresis on a 0.7 % agarose gel and transferred to a Hybond-N+ nylon membrane (Amersham Biosciences). Membrane bound digested genomic DNA was hybridized at 60˚C overnight with either the probe to the 5’ UTR or to the VEA1 CDS. Finally, the membranes were visualized by using CDP-Star Chemiflourescent detection system (GE Healthcare).
4
VEA1 Gene Expression Analysis
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A 1052-probe to the 5′ UTR of VEA1 was amplified from G217B ura5− genomic DNA using primer set OAS6659/OAS6660. A second 1013-bp probe to the VEA1 CDS was amplified from G217B ura5− genomic DNA using primer set OAS6663/OAS1969. In separate reactions, the resulting PCR fragments were coupled with an alkaline phosphatase enzyme using the Amersham Gene Images AlkPhos Direct Labelling and Detection System (GE Healthcare), according to the manufacturer’s protocol. Genomic DNA from the parental strain G217B ura5− and the mutant strains was isolated by phenol chloroform extraction. In duplicate, 15 µg of genomic DNA from both samples was digested with restriction enzyme BglII for 1 h at 37°C. Digested DNA was size separated by gel electrophoresis on a 0.7% agarose gel and transferred to a Hybond-N+ nylon membrane (Amersham Biosciences). Membrane-bound digested genomic DNA was hybridized at 60°C overnight with either the probe to the 5′ UTR or to the VEA1 CDS. Finally, the membranes were visualized by using CDP-Star Chemiflourescent detection system (GE Healthcare).
5
U. virens Gene Knockout and Complementation
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U. virens transformation was carried out as previously described (Chen et al., 2020b (link)). Briefly, about 1.2‐kb of the downstream and upstream flanking sequences of Uv1809 were ligated with the knockout vector pGKO. An approximately 3.5‐kb complementation fragment was cloned into the complementation vector pNeo3300III. The EHA105 Agrobacterium strain carrying the pGKO‐Uv1809, pNeo3300III‐Uv1809∆SP, or pNeo3300III‐Uv1809 vectors was transformed with the ATMT method. Transformed strains were confirmed by PCR analysis and Southern blot. Southern blot was performed using the Amersham Gene Images Alkphos Direct Labelling and Detection System (GE Healthcare, Little Chalfont, UK).
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