Genomic DNA digested with the appropriate restriction enzyme was run on 0.8% agarose gel overnight at 50 V. A specific probe was prepared by PCR amplification from genomic DNA. The probe was labeled using Amersham Gene Images AlkPhos Direct labeling and detection system (GE Healthcare). The labeled probe was immediately used to hybridize the DNA that was blotted on a nylon membrane. Chemifluorescent signal was generated and detected using CDP-Star as a substrate in conjugation with LAS-4000 luminescent image analyzer.
Amersham gene images alkphos direct labeling and detection system
Manufactured by GE Healthcare
Sourced in United Kingdom
The Amersham Gene Images AlkPhos Direct Labeling and Detection System is a laboratory equipment product designed for the direct detection of nucleic acids. It utilizes an alkaline phosphatase-based chemiluminescent detection method.
Automatically generated - may contain errors
Lab products found in correlation
Cl xposure film, by Thermo Fisher Scientific (4 mentions)
Zeta probe membrane, by Bio-Rad (4 mentions)
Stui enzyme, by New England Biolabs (2 mentions)
Rnase free dnase 1 treatment, by Takara Bio (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Trizol plus rna purification kit, by Thermo Fisher Scientific (1 mentions)
Gel purification kit, by Qiagen (1 mentions)
Hindiii, by New England Biolabs (1 mentions)
Ecori hf, by New England Biolabs (1 mentions)
Bamhi hf, by New England Biolabs (1 mentions)
Biodyne nylon transfer membranes, by Pall Corporation (1 mentions)
Las 3000, by Fujifilm (1 mentions)
Multi gauge version 3, by Fujifilm (1 mentions)
10 protocols using amersham gene images alkphos direct labeling and detection system
1
Southern Blot Analysis of Genomic DNA
2
Southern Blotting of Genomic DNA
3
Generating UvCGBP1 Knockout and Complementation Mutants
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To generate UvCGBP1 knockout mutants, 1.0 ~ 1.5-kb of upstream and downstream flanking sequences of UvCGBP1 were cloned into pGKO to construct the knockout vector pGKO-UvCGBP1. For complementation experiments, an approximately 4-kb fragment (containing 2-kb native promoter region and UvCGBP1 gene without termination codon) was ligated with the pNeo3300III vector. The EHA105 strain with the pGKO-UvCGBP1 and pNeo3300III-UvCGBP1 vectors were transformed with ATMT method by co-culture with conidia of the WT or ΔUvCGBP1-33, respectively. Transformed strains were confirmed by RT-PCR and Southern blot analysis. Based on the protocol of Amersham Gene Images Alkphos Direct Labeling and Detection System (GE Healthcare, UK), southern blot analysis was conducted. Gene deletion and complementation of other UvCGBP1 target genes were performed using similar methods described above.
4
Genomic DNA Extraction and Expression Analysis
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Genomic DNA was extracted using a CTAB method [18 (link)]. Southern blot analysis was conducted based on protocol of the Amersham Gene Images Alkphos Direct Labeling and Detection System (GE Healthcare, Amersham, UK). The probe DNA was amplified from the genomic DNA of the wild-type strain with labeled primers Probe-s/Probe-a (Table S1 ).
The Arabidopsis leaves inoculated with C. higginsianum at 24, 48 and 72 hpi were collected, flash frozen in liquid nitrogen, and stored at −80 °C. RNA isolation was carried out using the TRIzol® Plus RNA Purification Kit (Invitrogen, Carlsbad, CA, USA), and potential DNA contamination was removed by DNase I treatment (RNase Free) (TaKaRa, Dalian, China) following the manufacturer’s instructions. First-strand cDNA was synthesized with the Revert Aid first strand cDNA Synthesis kit (Fermentas, St. Leon-Rot, Germany) following the manufacturer’s instructions. RT-PCR was used for confirmation of deletion and complementation mutants following the methods in previous work [23 (link)]. Expressions of ChHxt genes at different infection stages were analyzed by Real Time-PCR, and α-tubulin (ChαTUB, CH63R_12878) was used as the reference gene. The primers used in RT-qPCR assays are listed inTable S1 . Each experiment was repeated three times.
The Arabidopsis leaves inoculated with C. higginsianum at 24, 48 and 72 hpi were collected, flash frozen in liquid nitrogen, and stored at −80 °C. RNA isolation was carried out using the TRIzol® Plus RNA Purification Kit (Invitrogen, Carlsbad, CA, USA), and potential DNA contamination was removed by DNase I treatment (RNase Free) (TaKaRa, Dalian, China) following the manufacturer’s instructions. First-strand cDNA was synthesized with the Revert Aid first strand cDNA Synthesis kit (Fermentas, St. Leon-Rot, Germany) following the manufacturer’s instructions. RT-PCR was used for confirmation of deletion and complementation mutants following the methods in previous work [23 (link)]. Expressions of ChHxt genes at different infection stages were analyzed by Real Time-PCR, and α-tubulin (ChαTUB, CH63R_12878) was used as the reference gene. The primers used in RT-qPCR assays are listed in
5
Southern Blot Analysis of Mutant Genomic DNA
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RFLP Analysis with RG57 Probe
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DNA extractions and subsequent restriction fragment length polymorphism (RFLP) analysis with the RG57 DNA probe were performed using a method modified from Goodwin et al. [23] (link). Southern blot analysis was conducted using the Amersham gene images AlkPhos direct labeling and detection system (GE Healthcare) according to the manufacturer's instructions. The US-1 (SA960008) reference isolate was used in RG57 analyses. Presence or absence of known fingerprint fragments was scored visually.
7
Southern Blot Analysis of Aspergillus Genomic DNA
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Genomic DNA from A. parasiticus NR-1, JW12, and JW13 was isolated from frozen fungal mycelium using the phenol-chloroform method previously described [45 (link)]. Southern hybridization analysis was performed as previously described [46 (link)] with minor modifications. Briefly, 2.5 μg genomic DNA was digested with 20 U of BamHI-HF, EcoRI-HF, or HindIII at 37 °C for 16 h (NEB, Ipswich, MA, USA). Digested DNA was separated on a 0.8% agarose gel and transferred onto a Nytran® SuPerCharge membrane (Schleicher and Schell, Inc., Keene, NH, USA) by capillary transfer. Membranes were UV cross-linked (120,000 μJ /cm2) in a UV Stratalinker® 2400 (Stratagene, Inc., La Jolla, CA, USA). A 600-bp atfB probe was generated by PCR (5′-ATGTCGGTGGACCAAACCCT-3′, forward primer; 5′-TCGCCTTTCTTGCTGGATAC-3′, reverse primer) (Figure S1 ). PCR products were purified with a Qiagen Gel Purification kit (Qiagen, Valencia, CA, USA). Probe labeling, hybridization, the washing of membranes at 55 °C, and detection were performed using an Amersham Gene Images AlkPhos Direct Labeling and Detection System (GE Healthcare, Pittsburg, PA, USA) according to manufacturer’s instructions.
8
Southern Blotting of Genomic DNA
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Southern blotting was performed as previously described in detail9 (link). Genomic DNA was digested with StuI enzyme (R0187L, New England Biolabs) and separated on a 0.7% Tris-borate-EDTA (TBE) agarose gel. The DNA in the gel was depurinated in 0.25 M HCl, denatured in a bath of 0.5 M NaOH, 1M NaCl, neutralized in a bath of 1.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, and finally transferred onto a Zeta-probe membrane (1620159, Bio-Rad) overnight using the alkaline transfer protocol given in the manual accompanying the membrane. On the next day, the membrane was gently washed with saline-sodium citrate (2xSSC: 0.3 M NaCl, 0.03 M sodium citrate), dried with paper towel, and UV cross-linked twice using the Stratalinker1800 (Stratagene). For probe generation, the AphVIII gene on CIB1 was amplified using primers oMJ588 and oMJ589 (Supplementary Table 1 ). The PCR product was purified and labeled according to the protocol of Amersham Gene Images AlkPhos Direct Labeling and Detection System (RPN3690, GE Healthcare). The membrane was hybridized at 60°C overnight with10 ng probe/mL hybridization buffer. On the next day, the membrane was washed with primary and secondary wash buffers and then visualized using a CL-XPosure film (34093, Thermo Fisher).
9
Northern Blot Analysis of RNA
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Fifteen micrograms of total RNA was electrophoresed on a 1.2% agarose gel and blotted onto Biodyne Nylon Transfer Membranes (Pall Corp., NY). RNA was cross-linked using ultraviolet radiation (1,200 × 100 μJ/cm2; Spectrolinker XL-1500, Spectronics Corp., NY). Ten micrograms of DNA were labeled using Amersham Gene Images AlkPhos Direct Labeling and Detection System (GE Healthcare, Little Chalfont, United Kingdom) and added to hybridization buffer for 20 h at 55 °C. Chemiluminescence was detected for 2,000 s with a lumino-image analyzer LAS3000 (Fujifilm Corp.) after washing the membranes, and analyzed with Multi Gauge version 3.0 software (Fujifilm Corp.). As a loading control, the gels were also stained by ethidium bromide.
10
Southern Blot Analysis of Genomic DNA
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Genomic DNA was extracted from the mutants and their parental strain by phenol–chloroform extraction as described previously (Zhang et al., 2014 (link)). Five micrograms was digested overnight with 50 units of StuI. The genomic DNA digests were run at 4°C on a 0.7% TRIS-borate-EDTA (TBE) gel for 17 h at 30 mV, and then for two more hours at 100 mV. The gel was depurinated in 0.25 m HCl for 15 min, and transferred onto a Zeta Probe membrane (Bio-Rad, http://www.bio-rad.com/ ) in 0.4 m NaOH for 24 h. The blot was rinsed in 2× saline sodium citrate (SSC) buffer and then crosslinked using a UV Stratalinker. Hybridization and signal detection were done following the Amersham Gene Images AlkPhos Direct Labeling and Detection System (GE Healthcare, http://www3.gehealthcare.com/ ) with the following changes: the primary wash was performed for 3 h and the secondary one for 40 min. The blot was exposed overnight onto CL-XPosure film (Thermo Scientific, http://www.thermoscientific.com/en/home.html ) and scanned.
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