The DNAs digested with SmaI and separated by the PFGE were transferred to nylon membranes (Amersham Hybond™-N; GE Healthcare) by vacuum blotting. The blotted membranes were prehybridized overnight in DIG Easy Hyb (Roche) at 42°C. The digoxigenin (DIG)-labeled probes specific to IS1216V, ermB and cat were prepared using DIG PCR Probe Synthesis Kit (Roche). The membrane was hybridized with the specific probe overnight in DIG Easy Hyb (Roche) at 42°C. The detection of hybridization was performed with an anti-DIG antibody conjugated to alkaline phosphatase, and CSPD (Roche) was used as a substrate according to the manufacturer’s instructions.
Dig easy hyb
Manufactured by Roche
Sourced in United States, Switzerland, Germany
The DIG Easy Hyb is a laboratory equipment product designed for hybridization applications. It provides a consistent and controlled environment for various hybridization procedures. The core function of the DIG Easy Hyb is to facilitate the hybridization of nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (18 mentions)
Pcr dig probe synthesis kit, by Roche (11 mentions)
Dig luminescent detection kit, by Roche (10 mentions)
Positively charged nylon membrane, by Roche (8 mentions)
Dig wash and block buffer set, by Roche (7 mentions)
Hybond n, by GE Healthcare (6 mentions)
Hybond n nylon membrane, by GE Healthcare (4 mentions)
Masterpure yeast dna purification kit, by Illumina (3 mentions)
Anti digoxigenin ap fab fragment, by Roche (3 mentions)
Las 4000, by Cytiva (3 mentions)
Dig dna labeling mix, by Roche (3 mentions)
Blocking one, by Nacalai Tesque (3 mentions)
Nylon membrane, by Roche (3 mentions)
Cdp star reagent, by Roche (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Cdp star, by Roche (3 mentions)
Cdp star ready to use kit, by Roche (2 mentions)
Taq dna polymerase, by New England Biolabs (2 mentions)
Chemidoc mp, by Bio-Rad (2 mentions)
Cdp star chemiluminescent substrate, by Roche (2 mentions)
59 protocols using dig easy hyb
1
Detecting Antibiotic Resistance Genes
2
Telomere Length Analysis by Southern Blot
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DNA samples, extracted from leukocyte, were digested with restriction enzymes Hinf and Risa (1 μg each; Roche, Mannheim, Germany). Sixteen DNA samples (17 μg each), high and low DNA control (17 μg each), and 2 DNA ladders (l kb) were resolved on a 0.8% agarose gel (20 cm×20 cm) at 100 V in 1×TAE buffer. After 8 hr, the DNA was depurinated for approximately 5–10 min in 0.25 bM HCl solution until the bromophenol blue stain changed its color to yellow, denatured for 15 min ×2 in 0.5-M NaOH, 1.5-M NaCl, and neutralized for 15 min ×2 in 0.5-M Tris, 3-M NaCl, pH 7.5. The DNA was transferred overnight to a positively charged nylon membrane using 20×saline-sodium citrate (SSC). After Southern blot transfer, the transferred DNA was fixed on the wet blotting membrane by ultraviolet-crosslinking (120 mJ) and membranes were baked at 65°C for 4 hr. The membranes were prehybridized at 42°C with prewarmed DIG Easy Hyb for 4 hr (Roche, Mannheim, Germany) and hybridized overnight at 42°C with prewarmed DIG Easy Hyb and a 1-μL telomere probe (Roche, Mannheim, Germany). The membranes were washed twice with 2×SSC for 5 min and twice with 0.2×SSC for 20 min at 50°C. After the membranes were washed, telomeric DNA was detected by the chemiluminescence detection procedure (Roche, Mannheim, Germany) and exposed on a radiograph.
3
Northern Blot Analysis of RNA Transcripts
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Twenty micrograms of total RNA extracted from HK1651 harvested during the early-exponential phases was electrophoresed on a TBE-Urea (9M)-acrylamide (8%) gel. The separated RNA was transferred onto a positively charged nylon membrane (Roche Diagnostics, Basel, Switzerland) in 20× SSC. The membrane was baked for 2 h at 80°C. After prehybridization using DIG Easy Hyb (Roche Diagnostics), the membrane was hybridized with DIG Easy Hyb containing a 25 pmol of single-stranded DNA probe (Supplementary Table S1 ) labelled with digoxigenin using DIG Oligonucleotide Tailing Kit, 2nd generation (Roche Diagnostics) for 18 h at 45°C. After hybridization, the membrane was washed and blocked using DIG Wash and Block Buffer Set (Roche Diagnostics), and then the hybridization signals were detected using DIG Luminescent Detection Kit (Roche Diagnostics) and ChemiDoc™ XRS+ (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
4
Aspergillus fumigatus DNA Manipulation
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Manipulation of DNA was carried out according to standard procedures (48 ). Sequence information was obtained from the Aspergillus Genome Database (AspGD; http://www.aspergillusgenome.org ) (49 (link), 50 (link)). Chromosomal DNA of A. fumigatus was isolated using a MasterPure Yeast DNA purification kit (Epicentre Biotechnologies). For Southern blot analysis, chromosomal DNA of A. fumigatus was digested with the indicated restriction enzymes (New England Biolabs). DNA fragments were separated in an agarose gel and blotted onto Hybond N+ nylon membranes (GE Healthcare Bio-Sciences). Labeling of DNA probes, hybridization, and detection of DNA-DNA hybrids were performed using digoxigenin (DIG) labeling mix, DIG Easy Hyb, and a CDP-Star ready-to-use kit (Roche Applied Science), respectively, according to the manufacturer’s recommendations.
5
Sensitive non-radioactive miRNA detection
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A sensitive non-radioactive northern blot method to detect miRNAs was performed as above (42 (link)). The northern blot analysis was carried out using microRNA Detection Probes with DIG-labeling (Exiqon) and a chemiluminescent reaction by enzyme-immunoassay. Firstly, equal amount of in vitro processing RNA substrate was dissolved in TBE-Urea Sample Buffer (Invitrogen), and heated at 95°C for 5 min then rapidly cooled on ice. The RNA was then loaded onto a denaturing 15% polyacrylmide–7.5 M urea gel and transferred electrophoretically to Hybond Nmembranes (Amersham Pharmacia Biotech). The membrane was dried at 80°C for 10 min and then pre-hybridized in 10 ml of pre-heated DIG Easy Hyb (Roche) at 45°C for 1 h. The membrane was hybridized overnight with the hsa-miR-21 DIG labeled LNA-DNA probe (Exiqon) at the concentration of 5 pmol/ml in hybridization oven at 53°C. After hybridization and washing, the membrane was detected with DIG Luminescent Detection Kit (Roche) according to the manufacturer's instructions. Lastly, the membrane was exposed to Amersham Hyperfilm ECL (GE Healthcare Life Sciences, Piscataway, NJ). The bar graphs corresponding to the northern blots were generated through densitometric analysis with ImageJ software.
6
Northern Blot Analysis of Total RNA
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10 µg of total RNAs were separated on 1% agarose gel containing 3% formaldehyde, rinsed in distilled water, denatured in 0.02 N NaOH for 20 min, equilibrated in 20× SSC, and blotted on a nylon membrane following standard procedure. DIG-labeled probes were hybridized in DIG Easy Hyb (Roche, #11603558001) at a concentration of 0.1 µg/mL, and hybridized probes were detected with alkaline phosphatase-conjugated anti-DIG antibodies (Roche, #11093274910) and CDP-star (Roche, #CDP-RO). Chemiluminescence signals were detected with the ChemiDoc Touch Imaging System (Bio-Rad) and quantified using the ImageJ software.
7
Determination of Thiolation Status in mt-tRNAs
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Determination of the thiolation status of mt-tRNAs was carried out as previously described [18 (link), 79 (link)]. Briefly, a volume of approximately 500 μl of worm pellet obtained from mixed-stage populations of liquid-cultured worms was processed using the “Isolation of small and large RNA” NucleoSpin miRNA kit (Macherey-Nagel) and the manufacturer’s instructions. 10 to 20 μg of total small RNA were run on 10% polyacrylamide/8 M urea gels with or without 0.01 mg/ml APM ([p-(N-acrylamino)-phenyl]mercuric chloride). APM was synthesized and kindly provided by Prof. Stephane Vincent [30 (link)]. Gels were run at 200 V for 3 h at 4°C and electroblotted onto positively charged nylon membranes. The RNA was crosslinked to the membrane by exposure to UV light for 2 min and baking at 80°C for 45 min. Pre-hybridization and hybridization were performed with Dig Easy Hyb (Roche) according to the manufacturer’s instructions. mt-tRNAs were detected with specific DIG-labeled synthetic oligodeoxynucleotides (S2 Table ) used at 10 nM final concentration. The blots were detected using the DIG luminescent detection CDP-Star kit (Roche) according to the manufacturer´s instructions.
8
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).
9
miRNA Detection Using Polyacrylamide Gels
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In brief, 15% polyacrylamide-8M urea gels were used to isolate the low-molecular-weight
RNA-enriched fraction. Band sizes were estimated by comparing them to a 10 bp DNA Marker
(NEB; Ipswich, MA). Gels were transferred to Hybond-N+ membranes (GE Healthcare; Uppsala,
Sweden) by capillary transfer. After cross-linking, membranes were placed in DIG-easy Hyb
(Roche; Indianapolis, IN). DIG-labeled riboprobes were synthesized using an mirVana miRNA
probe construction kit (Ambion) using the following DNA oligonucleotide as a template:
miR-mMC4R sense probe (for guide strand siRNA detection),
5′-AAAAAGTTGCTCATAGCATCCCCTGTCTC-3′. After hybridization at room temperature overnight,
membranes were washed twice at 37°C for 15 min in 0.1 × SSC, 0.1% SDS. Probe detection was
performed using a DIG Luminescent Detection Kit (Roche) according to the manufacturer’s
protocol.
Stripping of the riboprobe was performed at 68°C for 30 min in 0.1% SDS, followed by
washing in 2× SSC at room temperature. Then membranes were reprobed with miR-16 riboprobe
(Ambion) in the same manner as described above.
RNA-enriched fraction. Band sizes were estimated by comparing them to a 10 bp DNA Marker
(NEB; Ipswich, MA). Gels were transferred to Hybond-N+ membranes (GE Healthcare; Uppsala,
Sweden) by capillary transfer. After cross-linking, membranes were placed in DIG-easy Hyb
(Roche; Indianapolis, IN). DIG-labeled riboprobes were synthesized using an mirVana miRNA
probe construction kit (Ambion) using the following DNA oligonucleotide as a template:
miR-mMC4R sense probe (for guide strand siRNA detection),
5′-AAAAAGTTGCTCATAGCATCCCCTGTCTC-3′. After hybridization at room temperature overnight,
membranes were washed twice at 37°C for 15 min in 0.1 × SSC, 0.1% SDS. Probe detection was
performed using a DIG Luminescent Detection Kit (Roche) according to the manufacturer’s
protocol.
Stripping of the riboprobe was performed at 68°C for 30 min in 0.1% SDS, followed by
washing in 2× SSC at room temperature. Then membranes were reprobed with miR-16 riboprobe
(Ambion) in the same manner as described above.
10
Detection of Outer Membrane Protein via Northern Blot
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