Total RNA (8–20 μg) from 21 hpf embryos and adults were isolated using RNAiso Reagent (Takara) and electrophoresed in a denaturing 12% polyacrylamide gel and transferred onto a Hybond-N+ membrane (GE Healthcare). miRNA probes (Additional file 5 : Table S3) were DIG-labeled LNA-modified probes synthesized by Exiqon (Denmark). Hybridization and washing were performed as described by Válóczi [36 (link)]. Signals were detected using CDP-Star chemiluminescent substrate (Roche), and the blot was exposed to the Lumi-Imager F1 Workstation. To control equal loading, blots were hybridized with a DIG-labeled probe against U6 snRNA.
Cdp star chemiluminescent substrate
Manufactured by Roche
Sourced in Switzerland
CDP-Star is a chemiluminescent substrate used in various laboratory applications. It is a sensitive and stable reagent that generates a luminescent signal when used in conjunction with appropriate enzyme-labeled probes or antibodies. The core function of CDP-Star is to provide a detection mechanism for the visualization and quantification of target analytes in various assays.
Automatically generated - may contain errors
Lab products found in correlation
Anti digoxigenin ap fab fragment, by Roche (3 mentions)
Dig easy hyb granules, by Roche (2 mentions)
Taq dna polymerase, by New England Biolabs (2 mentions)
Dig easy hyb, by Roche (2 mentions)
Aatii, by Thermo Fisher Scientific (2 mentions)
Pcr dig dna labelling mix, by Roche (2 mentions)
Hybond n, by GE Healthcare (2 mentions)
Chemidoc mp, by Bio-Rad (2 mentions)
Zeta probe gt, by Bio-Rad (2 mentions)
Tripure reagent, by Aidlab (2 mentions)
Dig dna labeling mix, by Roche (2 mentions)
Mirna probes, by Qiagen (1 mentions)
Lumi imager f1 workstation, by Roche (1 mentions)
Hybond n membrane, by GE Healthcare (1 mentions)
Rnaiso reagent, by Takara Bio (1 mentions)
Zeta probe gt membrane, by Bio-Rad (1 mentions)
Zeta probe, by Bio-Rad (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Urea, by Fujifilm (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
16 protocols using cdp star chemiluminescent substrate
1
Detecting miRNA Expression in Embryos
2
Quantification of small interfering RNAs
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For sisRNA analysis, 10–30 µg of total RNA was separated on an 8% denaturing polyacrylamide gel (8 M urea, 1× TBE buffer). RNA was transferred by electrophoresis onto a nylon membrane (Zeta-Probe GT membrane; Bio-Rad Laboratories). For ASTR analysis, 8–10 µg of total RNA was separated on a 0.8% agarose/formaldehyde gel. RNA was transferred by capillary action onto a nylon membrane (Zeta-Probe GT membrane; Bio-Rad Laboratories). RNA was then UV cross-linked to the membranes, prehybridized with salmon sperm DNA, and hybridized overnight (14–16 h) with probes in DIG Easy Hyb Granules (Roche) at 51°C (for mbt sisRNA) or 42°C (for all other sisRNAs). DIG-labeled DNA probes were synthesized by PCR with genomic DNA as the template and purified before using. After hybridization, the membranes were rinsed once with 2× SSC, followed by one wash with 2× SSC and 0.1% SDS, and two washes with 0.1× SSC and 0.1% SDS. Detection was performed using the CDP-Star chemiluminescent substrate (Roche) and exposed on x-ray films.
3
Verifying Homologous Gene Deletion in Fungi
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To confirm the homologous integration of the deletion construct into the genome, gel-purified gDNA from potential Δpks1 and Δku80_Δpks1 transformants and their parental strains were digested overnight by PstI (Thermo). gDNA of Δku80 strains was digested overnight with AatII (Thermo). Digested gDNA was loaded on a 0.8% agarose gel, separated by electrophoresis, and transferred to a nylon membrane Hybond N+ (GE Healthcare). The probes for the upstream regions of pks1 (oligonucleotides 9 and 10) and ku80 (oligonucleotides 24 and 25) deletion constructs were synthetized using PCR DIG DNA Labelling Mix (Roche) and Taq DNA Polymerase (NEB). Probes were hybridized overnight in DIG Easy Hyb (Roche) at 42°C and detection was performed by chemiluminescence using anti-Digoxigenin-AP Fab fragments (Roche) and CDP-Star chemiluminescent substrate (Roche) in a ChemiDoc MP (Bio-Rad).
4
Verifying Homologous Gene Deletion in Fungi
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To confirm the homologous integration of the deletion construct into the genome, gel-purified gDNA from potential Δpks1 and Δku80_Δpks1 transformants and their parental strains were digested overnight by PstI (Thermo). gDNA of Δku80 strains was digested overnight with AatII (Thermo). Digested gDNA was loaded on a 0.8% agarose gel, separated by electrophoresis, and transferred to a nylon membrane Hybond N+ (GE Healthcare). The probes for the upstream regions of pks1 (oligonucleotides 9 and 10) and ku80 (oligonucleotides 24 and 25) deletion constructs were synthetized using PCR DIG DNA Labelling Mix (Roche) and Taq DNA Polymerase (NEB). Probes were hybridized overnight in DIG Easy Hyb (Roche) at 42°C and detection was performed by chemiluminescence using anti-Digoxigenin-AP Fab fragments (Roche) and CDP-Star chemiluminescent substrate (Roche) in a ChemiDoc MP (Bio-Rad).
5
Northern Blot Analysis of RNA
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Up to 5 μg of RNA was separated on an 8% polyacrylamide–8 M urea gel and transferred onto a nylon membrane (Zeta Probe GT; Bio-Rad). RNA was probed with dsDNA labeled with digoxigenin (DIG)-dUTP in hybridization buffer and detected using an anti-DIG antibody conjugated with alkaline phosphatase and CDP-Star chemiluminescent substrate (Roche).
6
Northern Blot Analysis of sisR-1 and ASTR
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Northern blotting was performed as described previously5 (link). DIG-labeled DNA probes were made by PCR using genomic DNA as the template. To detect sisR-1, RNA was run on an 8% polyacrylamide gel (8 M urea, 1× TBE buffer), and transferred onto a nylon membrane by electrophoresis. To detect, ASTR, RNA was run on a 0.8% agarose/formaldehyde gel and transferred onto a nylon membrane by capillary action. RNA was UV crosslinked to the membrane, pre-hybridized in salmon sperm DNA, and hybridized with probes in DIG Easy Hyb Granules (Roche) at 42 °C overnight. Next day, the membranes were washed once with 2× SSC and 0.1% SDS, and twice with 0.1× SSC and 0.1% SDS, followed by detection with the CDP-Star chemiluminescent substrate (Roche).
7
Southern Blot Analysis of DNA Fragments
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A DNA fragment between 0.7 and 0.1 kb from the upstream or downstream region of every gene was amplified and used as a probe for Southern blot analysis (Fig. S1 ). The probes were labelled with the HighPrime Kit (Roche Applied Sciences, Almere, the Netherlands). About 15 μg of gDNA, previously digested with suitable restriction enzymes, was separated by electrophoresis on an 0.8% agarose gel. The gel was equilibrated in 20× saline–sodium citrate (SSC) buffer (3 M NaCl; 0.3 M C6H5Na3O7; pH 7) and the DNA was transferred overnight to a positively charged nylon membrane (Zeta‐Probe; Bio‐Rad, Munchen, Germany). Subsequently, the membrane was incubated overnight with the labelled probe(s). For detection, the membrane was treated with anti‐DIG Fab fragment alkaline phosphatase and the CDP‐Star chemiluminescent substrate (Roche Applied Sciences). The signal was measured using a Lumi‐Imager (Fujifilm LAS‐4000, Fujifilm Co. Ltd, Tokio, Japan).
8
Monitoring mRNA Expression under Mitomycin C Stress
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MG1655-Sp5 cells were grown in LB broth at 37 °C until the OD660 reached 0.4, and harvested at 0, 15, 30, 60, 90, and 120 min after 2.0 µg/mL MMC addition. Total RNA was isolated and purified as previously described [45 (link)]. Total RNA (2.0 µg) was electrophoresed on a 6% polyacrylamide gel (FUJIFILM Wako Pure Chemical, Osaka, Japan) containing 7 M urea (FUJIFILM Wako Pure Chemical, Osaka, Japan), followed by northern blotting. The digoxigenin (Dig)-labeled oligo-probe YO-597 or YO-592 (eurofins, Tokyo, Japan) was used for hokW mRNA or sokW RNA. After hybridization, the membranes were probed with anti-digoxigenin-AP Fab fragments (Roche, Basel, Switzerland), and RNAs were detected using CDP-Star chemiluminescent substrate (Roche, Basel, Switzerland) and the C-DiGit Blot Scanner (LI-COR, Lincoln, NE, USA).
9
Ganoderma lucidum Gene Expression Analysis
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For evaluating the gene expression of SQS and LS at the mRNA level, the corresponding DNA fragments were cloned from G. lucidum BCRC 3611118 (link), 54 (link), and northern blotting was performed using standard procedures. Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract fungal total RNA per the manufacturer’s protocol. The DNA probe for the SQS gene was amplified by PCR using the primers glssF263 (5′-TGGACACGATCGAAGATGACATGAC-3′) and glssR1492 (5′-GCCATCGTTTGTGGGATCGCACAGAA-3′), with the incorporation of digoxigenin-11-dUTP (Roche Applied Science). The primers gllsF1292 (5′-CGGCGTATCGGCACCAGACGAA-3′) and gllsR2105 (5′-TTCGGGTACGATATCGCGACGTTC-3′) were used to amplify the DNA probe for the LS coding region. Immunological detection of each northern blot was carried out using CDP-Star® chemiluminescent substrate according to the manufacturer’s instructions (Roche Applied Science). All experiments were conducted at least three times.
10
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.
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