The DNA-binding activity of PcDp1 was analyzed by using electrophoretic mobility shift assay (EMSA). The specific wild probe sequences (WE2Fsite-F and -R) and mutant probe sequences (ME2Fsite-F and -R) of PcDp1 were synthesized and are listed in Table 1 . The complementary sequence probe was incubated at 95 °C for 10 min in a 1:1 ratio, and then cooled slowly to 15–25 °C to form the two-stranded DNA probes. The two double-stranded DNA probes were labeled with biotin according to the protocols described in DIG Oligonucleotide 3′ End Labeling Kit (Roche Diagnostics, Germany). In the EMSA, biotin-labeled DNA probes were successively incubated with the purified rPcDp1 protein at room temperature for 15 min and on ice for 25 min in the presence of binding buffer from EMSA/gel-shift kit (Roche Diagnostics, Germany). For competition experiment, the purified rPcDp1 protein was re-incubated with the wild probes (twice) for 30 min before addition of the biotin-labeled probes. After the binding reactions, DNA-protein complexes were resolved by electrophoresis in 6% native acrylamide gel and transferred to a positively charged nylon membrane (Osmonics, USA). The membrane was immediately cross-linked with UV-light for 10 min. Then the membrane was fixed in a film cassette and was visualized by exposure to X-ray film in a dark room at room temperature for 15–25 min.
Dig oligonucleotide 3 end labeling kit
Manufactured by Roche
Sourced in United States, Switzerland
The DIG Oligonucleotide 3'-End Labeling Kit is a laboratory tool used for the labeling of oligonucleotides at the 3' end. The kit provides the necessary reagents and protocols to perform this labeling process.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (3 mentions)
Dig nucleic acid detection kit, by Roche (3 mentions)
Semi dry electroblotting, by Bio-Rad (2 mentions)
Ultrahyb hybridization buffer, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Northern max gly kit, by Thermo Fisher Scientific (2 mentions)
Cdp star, by Roche (2 mentions)
Mircury lna probe, by Qiagen (1 mentions)
Eclipse 800 microscope, by Nikon (1 mentions)
Bm purple ap substrate, by Roche (1 mentions)
Hybond n, by Cytiva (1 mentions)
Cdp star reagent, by Roche (1 mentions)
Positively charged nylon membrane, by Roche (1 mentions)
Anti dig fab fragment, by Roche (1 mentions)
Cdp star substrate, by Roche (1 mentions)
Amershan hybond n, by GE Healthcare (1 mentions)
Las 4000, by Cytiva (1 mentions)
Amersham hybond n, by GE Healthcare (1 mentions)
Trans blot sd, by Bio-Rad (1 mentions)
Dig high prime dna labeling and detection starter kit 2, by Roche (1 mentions)
32 protocols using dig oligonucleotide 3 end labeling kit
1
Analyzing the DNA-Binding Activity of PcDp1 Using EMSA
2
Localization of miR-183 in Zebrafish
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA probes against miR-183 were hybridized to fish fixed at 120 hpf. Hybridization detection was via alkaline phosphatase (AP)-conjugated anti-digoxigenin (Roche, Indianapolis, IN, USA), followed by the NBT/BCIP color reaction. Whole-mount in situ hybridization was performed using locked nucleic acid (LNA) probes for miR-183 labeled with digoxigenin (DIG Oligonucleotide 3′-End Labeling Kit; Roche). RNA probes for in situ hybridization were synthesized using Taq polymerase. LNA probes (miRCURY LNA probes) were purchased from Exiqon (Vedbaek, Denmark) or custom synthesized (Integrated DNA Technologies, Coralville, IA, USA) by incorporating LNA modifications at every third nucleotide position from the 5′ end. LNA probes are antisense to miR-183 (5′-CAGTGAATTCTACCAGTGCCATA). Briefly, fixed tissues were defatted with ethanol, digested with 10 µg/mL Proteinase K/PBT, hybridized with 12 pmol labeled LNA probe, washed, and digested with RNase A. Labeled LNA probe was detected using AP conjugated sheep anti-DIG Fab fragment and BM Purple AP Substrate (Roche). Tissues were whole mounted in glycerol and imaged using light microscopy using a Nikon Eclipse 800 micro-scope. A minimum of three samples were prepared for each condition.
3
Locked Nucleic Acid Probe Detection of miR-207
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The locked nucleic acid (LNA) probe for miR-207 labeled with digoxigenin (DIG Oligonucleotide 3'-End Labeling Kit; Roche, Stockholm, Sweden) were purchased from Exiqon Co (Vedbaek, Denmark). U6 RNA was detected using a DIG-labeled U6 DNA probe. Northern blotting was performed as described.31 (link) Total RNA from cochleas was resolved by 15% denaturing polyacrylamide gel electrophoresis and transferred by electroblotting to membranes. Blots were pre-hybridized and hybridized by overnight incubation in buffer containing 0.2 nM DIG-labeled probes at 52 °C for miR-207 LNA probe or 25 °C for the U6 DNA probe. Blots were stringently washed twice in SDS for 30 min at 52 °C for miR-207 LNA probe or 25 °C for the U6 DNA probe, rinsed in wash buffer, and incubated in block buffer for 30 min. Subsequently, blots were incubated with anti-DIG-AP Fab fragment in block buffer for 1 h, washed in wash buffer and detection buffer separately. Anti-DIG-AP was detected using CDP-star chemiluminescent substrate for alkaline phosphatase (AP). Blots were stripped by incubation at 70 °C in 0.1 × SSC containing 1% SDS and probed up to three times.
4
DNA-binding Assay with EMSA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA-binding tests were performed by electrophoretic mobility shift assay (EMSA). The DNA fragments used for EMSA were amplified by PCR using the primers listed in Table 1 and S. filipinensis genomic DNA as the template, sequenced to confirm the absence of any mutations, and then labeled at both ends with digoxigenin (DIG) using a DIG oligonucleotide 3′-end labeling kit (Roche Applied Science) (2nd generation).
A standard binding reaction mixture contained 0.005 ng/μl labeled DNA probe, 100 mM HEPES (pH 7.6), 10 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT), 1% Tween 20, 7.8 mM glutathione, 40 μg/ml poly[d(I·C)], and 5% glycerol in a 10-μl final volume. Reaction mixtures were incubated at 30°C for 10 min and then loaded onto 5% polyacrylamide (29:1) native gels in 0.5× Tris-borate-EDTA (TBE) buffer. After electrophoresis, DNA was electroblotted onto a nylon membrane (HyBond-N; Amersham Biosciences) in 0.5× TBE buffer. The DNA was fixed by UV cross-linking, detected with antidigoxigenin antibodies, and developed by chemiluminescence performed with CDP-Star reagent (Roche Applied Science).
When EMSAs were performed with the addition of antibiotics, antibiotic was added after 10 min preincubation of the probe with the protein (1 μM) and then further incubated for 10 min before loading onto polyacrylamide gels.
A standard binding reaction mixture contained 0.005 ng/μl labeled DNA probe, 100 mM HEPES (pH 7.6), 10 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT), 1% Tween 20, 7.8 mM glutathione, 40 μg/ml poly[d(I·C)], and 5% glycerol in a 10-μl final volume. Reaction mixtures were incubated at 30°C for 10 min and then loaded onto 5% polyacrylamide (29:1) native gels in 0.5× Tris-borate-EDTA (TBE) buffer. After electrophoresis, DNA was electroblotted onto a nylon membrane (HyBond-N; Amersham Biosciences) in 0.5× TBE buffer. The DNA was fixed by UV cross-linking, detected with antidigoxigenin antibodies, and developed by chemiluminescence performed with CDP-Star reagent (Roche Applied Science).
When EMSAs were performed with the addition of antibiotics, antibiotic was added after 10 min preincubation of the probe with the protein (1 μM) and then further incubated for 10 min before loading onto polyacrylamide gels.
5
Northern Blot Analysis of RNA Transcripts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Northern blotting was carried out according to Ref. [12] (link) with some modifications. Briefly, 20 μg RNA were resolved on denaturing polyacrylamide gels in 1X TBE, transferred onto a positively charged nylon membrane (Roche Diagnostics) using semi-dry electroblotting (Bio-Rad), and immobilized by UV irradiation (Stratagene Crosslinker 120 mJ/cm2). The membrane was pre-hybridized with hybridization buffer at 37 °C for 1 h, and then hybridized overnight at 37 °C with specific oligodeoxynucleotides. The probes were labeled with Digoxigenin using the DIG oligonucleotide 3′END labeling kit (Roche) and detected using the DIG Nucleic Acid Detection Kit (Roche). The following probes were used: RNU44: 5′-AGTTAGAGCTAATTAAGACCT-3′; tRNA-Ile, 5′-UGGUGGCCCGUACGGGGAUCGA-3′; U11: 5′-TCTTGATGTCGATTCCGCACGCAGAGCAATCGAGTTGCCC-3′; hY1: 5′-AAGGGGGGAAAGAGTAGAACA-3′. The probes were synthesized at the Johns Hopkins Genetic Resources Core Facility. The miRCURY locked nucleic acid (LNA) Digoxigenin-labeled probes for detection of miR-19b, miR-21, miR-23a, miR-24, miR-30c, miR-31, miR-99a and miR-191 were purchased from Exiqon.
6
Northern Blot Analysis of Coronavirus DI RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A Northern blot assay was performed as described previously [11 (link),13 (link)]. Briefly, HRT-18 cells in 35-mm dishes at ~80% confluency (~8 × 105 cells/dish) were infected with BCoV at a multiplicity of infection of five PFU per cell. Two hours post-infection (hpi), 3 µg of transcript was transfected into the HRT-18 cells. To detect the replication of BCoV DI RNA and sgmRNAs, the supernatant was collected at 48 hpt and then used to infect fresh HRT-18 cells (virus passage 1, VP1). Ten µg of TRIzol-extracted total cellular RNA at 48 hpi of VP1 was used for formaldehyde-agarose gel electrophoresis. To detect 18S rRNA, reporter-containing sgmRNA, and M sgmRNA, 10 µg of TRIzol-extracted total cellular RNA at 8 hpt (VP0) was used for formaldehyde-agarose gel electrophoresis. RNA was then transferred from the gel to Nytran membranes by vacuum blotting. The blots were probed with digoxigenin (DIG)-ddUTP labeled (DIG Oligonucleotide 3'-End Labeling kit; Roche Molecular Biochemicals) oligonucleotides: TGEV 8(+) (for reporter-containing BM25A, sBM25A, and sgmRNA), BCVN(+) (for M sgmRNA), and 18SrRNA(+) (for 18S rRNA). The detected RNA was visualized according to the procedure of the manufacturer.
7
Northern Blot RNA Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Northern blots were performed as previously described (49 (link)). A total of 20 μg of total RNA was used per lane and separated by electrophoresis on a formaldehyde 1.2% agarose gel. The RNA was subsequently transferred to a positively charged nylon membrane (Amershan Hybond N+, GE Healthcare) using electroblotting. Prior to hybridization the membranes were blocked using UltraHyb hybridization buffer (Ambion) for 1 h at 42°C. Hybridization was performed for 14–16 h using DIG-labelled probes (DIG Oligonucleotide 3′-End Labeling Kit, Roche). See Supplementary Table S3 for sequences of the used probes. Probes were detected using an AP-coupled Anti-DIG Fab fragments (Roche) followed by incubation with CDP-star substrate (Roche) and measurement with an LAS 4000 imaging system (Fujifilm, GE Healthcare).
8
RNA Extraction and Northern Blotting for G. arboreum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated from different tissues of G. arboreum using total plant RNA purification reagent (Invitrogen Cat No. 12322-012). RNA samples (30 μg) were diluted in 2X loading dye (95% formamide, 0.025% xylene cyanol, 0.025% bromophenol blue and 5mM EDTA) incubated at 65°C for 10 min for denaturation and placing on ice. 300pMol primer (complimentary to the probe) was loaded as positive control. Denatured RNA samples were loaded to 15% PAGE (Urea) and run at 100 V in BIO-RAD Mini protean tetrasystem United States. The gel was stained with ethidium bromide and visualized under UV light once the run was finished. RNA was transferred to the nylon membrane (Amersham HybondTM-N++, GE Healthcare) using semi dry blotter (Transblot SD BIO-RAD United States) at 15V for 1 h. RNA was cross linked to the membrane using UV Cross Linker (CL100o ultraviolet cross linker, UVP). The DNA Probe was synthesized using DIG Oligonucleotide 3′-End Labeling Kit (2nd generation from Roche Life Science, Catalog No.3353575910). The blot was prehybridised at 42°C for 3h and hybridized with probe at 42°C for 16 h. The blot was washed with 2X SSC shortly then twice with 2xSSC/0.1% SDS for 15mins and 30mins at 60°C. The blot was developed by chromogenic (NBT/BCIP-T) method using supplied protocol (Amin et al., 2011 (link); Zhang et al., 2013 (link)).
9
Northern Blot Analysis of CGMMV RNAs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from plants with Trizol (Invitrogen, USA) according to the manufacturer's instructions. For northern blot of CGMMV RNAs, a DNA probe targeting CGMMV CP was synthesized with primers (5′-GCTTACAATCCGATCACAC-3′ and 5′-ATTATCTATCTCAGCCCTAG-3′) and labeled with DIG according to the manufacturer's protocol (DIG Oligonucleotide 3′-end labeling Kit, Roche, USA). For northern blot of positive-stranded CGMMV RNAs in leaves, a sequence (5′-CAACACAGGACCGTTGAGGAAAGCGTAAAAACCCGCACCTGGGAATCTAGAATTAATATCTACGACAGACGAGGGTAACGCA-3′) was synthesized and labeled as DNA probe, and its complementary sequence was used for detecting negative-stranded CGMMV RNAs. Another sequence (5′-CATAGCTCTGAGCTTTAACTACACTAAAGTCAGTTATAGATAAATACTTAAGAATGGAAAAATAGTTAGGGAGCAACTTATC-3′) was used for detecting positive-stranded CGMMV RNAs in fruits, and its complementary sequence was used for detecting negative -stranded CGMMV RNAs in fruits. Pre-hybridization, hybridization and signal detection were done according to the protocol of the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, USA).
10
Northern Blot Analysis of TGEV DI RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ten micrograms of TRIzol-extracted total cellular RNA at 48 hpi of VP1 was used for electrophoresis in a formaldehyde-agarose gel. RNA was transferred from the gel to Nytran membrane by vacuum blotting, and the blots were probed with oligonucleotide TGEV 8(+) (for DI RNA), BCVN(+) (for N sgmRNA) or 18SrRNA(+) (for 18S rRNA), which was tailed with digoxigenin (DIG)-ddUTP using a DIG Oligonucleotide 3′-End Labeling kit (Roche Molecular Biochemicals). The RNA detected was visualized according to the manufacturer's recommended procedure.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!