Denhardt s

Manufactured by Merck Group

Sourced in United States

Denhardt's is a laboratory reagent used in molecular biology techniques. It serves as a blocking agent to reduce non-specific binding during nucleic acid hybridization assays, such as Northern blotting and Southern blotting. The core function of Denhardt's is to help improve the specificity and sensitivity of these hybridization-based analyses.

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Lab products found in correlation

Blocking reagent, by Roche (2 mentions) Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions) Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions) Oligo dt, by Merck Group (1 mentions) Salmon sperm dna, by Thermo Fisher Scientific (1 mentions) Sodium dodecyl sulfate (sds), by Roche (1 mentions) Immobilon ny nylon membrane, by Merck Group (1 mentions) Mgcl2, by Thermo Fisher Scientific (1 mentions) Formaldehyde, by Merck Group (1 mentions) Proteinase k, by Qiagen (1 mentions) Levamisole, by Merck Group (1 mentions) Nbt bcip solution, by Roche (1 mentions) Dig labeling kit, by Roche (1 mentions) Anti dig antibody, by Roche (1 mentions) Dextran sulfate, by Merck Group (1 mentions) Proteinase k, by Roche (1 mentions) Minelute gel extraction kit, by Qiagen (1 mentions) Formamide, by Merck Group (1 mentions) Ecorv, by Takara Bio (1 mentions) X ray film, by Merck Group (1 mentions)

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Denhardt's solution

4 protocols using denhardt s

Quantitative Analysis of DMPK and GAPDH mRNA Levels

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RNA was prepared by lysing 10 million cells in 2 mL of proteinase K solution (500 μg/mL proteinase K [QIAGEN]; 200 mM NaCl [Fisher Scientific]; 200 mM Tris-HCL, pH 7.5 [Wisent]; 1.5 mM MgCl2 [Fisher Scientific]; and 2% SDS [Roche]) for 30 min at 55°C. mRNA was then isolated with 60 mg of oligo(dT) (Sigma-Aldrich) using microcentrifuge spin columns (Ambion). Concentrated mRNA was quantified by absorbance at 260 nm on a NanoDrop 2000c (Thermo Scientific), and quality was verified on an agarose gel by controlling residual 28S/18S contamination. 3 μg of mRNA were separated on a 1% agarose gel containing MOPS (Laboratoire MAT) and 0.62 M formaldehyde (Sigma-Aldrich) and transferred to Immobilon-NY+ nylon membrane (Millipore) by downward alkaline blotting and hybridized with human ³²P-labeled DMPK or GAPDH oligonucleotide probes. Probes were generated by random priming (New England Biolabs) of 100 ng of DMPK cDNA fragments (BglII-SacI) or GAPDH cDNA PCR-amplified fragments (Table S3). Overnight hybridization was conducted at 65°C in hybridization buffer (1X SSPE [Sigma]; 2X Denhardt’s [Sigma]; 10% dextran sulfate [Fisher Scientific]; 1% SDS [Roche]; 100 μg/mL salmon sperm DNA [Ambion]; and probe 1 Mcpm/mL). Relative quantification of mRNA levels on scanned autoradiograms (600 dpi) was determined by densitometry using ImageJ 1.47 software.

Jauvin D., Chrétien J., Pandey S.K., Martineau L., Revillod L., Bassez G., Lachon A., McLeod A.R., Gourdon G., Wheeler T.M., Thornton C.A., Bennett C.F, & Puymirat J. (2017). Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice. Molecular Therapy. Nucleic Acids, 7, 465-474.

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In Situ Hybridization for Olfm4 Expression

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Olfm4 cDNA plasmid (IMAGE clone 9055739) was linearized and sense and antisense probes were made with DIG labeling kit according to the manufacturer’s instructions (Roche). Probes were purified using the Min-Elute Gel Extraction kit (Qiagen). Slides were deparaffinized, hydrated, and treated with 0.2N HCl for 15 minutes at room temperature. Tissues were treated with Proteinase K (30μg/mL, Roche) for 30 minutes at 37°C, post-fixed in 4% PFA for 10 minutes, acetylated for 10 minutes in 13.4% triethanolamine, 0.2M HCl and 2.5% acetic anhydride, incubated with hybridization buffer (50% deinonized formamide (Invitrogen), 10% dextran sulfate (Millipore), 2% Denhardts (Sigma), 1 mg/mL yeast tRNA, 0.2M NaCl, 0.1M Tris-HCl, pH 7.5, 0.1M phosphate buffer, pH 6.8, 5mM EDTA, pH 8 in DEPC-treated H₂O) for 1 hour, followed by incubation with Olfm4 probe diluted in hybridization buffer at 68°C overnight. Tissue sections were then washed, incubated in blocking solution (20% heat-inactivated serum, 0.02g/mL blocking reagent (Roche) in buffer (0.1M Tris-HCl, pH7.5, 0.15M NaCl, 0.1% Tween 20 in sterile H₂O) for 1 hour, and anti-DIG antibody (1:2500, Roche) overnight at 4°C. Slides were washed and developed with NBT/BCIP solution (1:100, Roche) in 0.1M Tris-HCl, pH9.5, 0.1M NaCl, 0.05M MgCl₂, 0.5mg/mL levamisole (Sigma) in sterile H₂O. Minimal signal was detected with Olfm4 sense probe control.

Carulli A.J., Keeley T.M., Demitrack E.S., Chung J., Maillard I, & Samuelson L.C. (2015). Notch receptor regulation of intestinal stem cell homeostasis and crypt regeneration. Developmental biology, 402(1), 98-108.

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Confirmation of Knocked-in Cells by Southern Blot

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Final confirmation of knocked-in cells was performed by southern blot analysis. Ten μg of the genomic DNA was digested with EcoRV (Takara Co., Japan), followed by purification. The DNA was electrophoresed on 0.8% agarose gel, and was then transferred on nitrocellulose membrane (Bio-Rad Co., USA). The membrane was washed with 2×saline-sodium citrate (SSC) buffer for 30 s. The DNA was cross-linked onto the membrane by using a UV crosslinker, followed by hybridization in the solution containing 5×saline-sodium phosphate EDTA (SSPE), 5×Denhardt’s (Sigma Chemical Co., USA), 1% sodium dodecyl sulfate (SDS) (w/v) and 50% formamide (w/v) (Sigma Chemical Co., USA) at 42°C for 15 h. A probe was prepared by using a 600 bp/SpeI-EcoRI DNA fragment containing the porcine β-casein exon 9, random labeling kit (Amersharm, Amersharm, England) and (α-32P) dCTP (110 TBq/mmol, Amersharm., England). A neo probe was also prepared by using 600 bp/PstI DNA fragment from pKJ2 plasmid. The concentration of the probe for hybridization was one million cpm/mL hybridization solution. After hybridization, the membrane was washed three times with 0.2% SSC and 0.1% SDS (w/v) at 68°C for 20 min, exposed on X-ray film (Sigma Chemical Co., St Louis, MO, USA) at −80°C for 4 days, and then developed.

Lee S.M., Kim J.W., Jeong Y.H., Kim S.E., Kim Y.J., Moon S.J., Lee J.H., Kim K.J., Kim M.K, & Kang M.J. (2014). Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into β-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein. Asian-Australasian Journal of Animal Sciences, 27(11), 1644-1651.

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Fluorescence In Situ Hybridization Protocol

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For fluorescence in situ hybridization (FISH), sections were incubated in 10 mM citrate buffer (pH 6) for 20 min at 85 °C, washed several times in 2× SSC (LONZA AccuGEN), and prehybridized for 2 h at 60 °C with hybridization buffer (50% formamide, 5× SSC, 5% dextran sulfate, 500 μg/mL DNA MB grade from sperm [Roche], 250 μg/mL t-RNA [Sigma Aldrich] and 1× Denhardt’s [Sigma Aldrich]). After heat treatment of FISH probes for 5 min at 85 °C, sections were incubated with probe in hybridization buffer (1:1000) overnight at 60 °C. Several washing steps (2× SSC for 10 min at RT, 2× SSC/50% formamide [AppliChem] for 15 min at 60 °C, 0.1× SSC/50% formamide for 15 min at 60 °C, 0.1× SSC for 15 min at 60 °C, and TN buffer [0.1 M Tris-HCl, 0.15 M NaCl; pH 7.4] for 5 min at RT) were performed before blocking solution (1% blocking reagent [Roche] in TN buffer) was added for 30 min at RT. After blocking, anti-Digoxigenin-POD (1:2000 in blocking solution; Roche) was added to sections for 2 h at RT. Sections were washed several times with TNT (0.1 M Tris-HCl, 0.15 M NaCl, 0.3% Triton X-100) and incubated in TSA-Plus Cyanine 3 System (Perkin Elmer) diluted in amplification solution (1:50) for 10 min in the dark at RT. After several washing steps with TNT, sections were further processed for immunohistochemistry.

Paul M.H., Choi M., Schlaudraff J., Deller T, & Del Turco D. (2019). Granule Cell Ensembles in Mouse Dentate Gyrus Rapidly Upregulate the Plasticity-Related Protein Synaptopodin after Exploration Behavior. Cerebral Cortex (New York, NY), 30(4), 2185-2198.

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