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Dna dot blot protocol

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The DNA Dot Blot Protocol is a laboratory technique used to detect and analyze the presence of specific DNA sequences in a sample. It involves the immobilization of DNA samples on a membrane, followed by hybridization with a labeled probe that binds to the target DNA sequence. The resulting signal can be used to quantify the amount of the target DNA present in the sample.

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Lab products found in correlation

Quick dna miniprep plus kit, by Zymo Research (3 mentions) Stripping buffer, by Thermo Fisher Scientific (2 mentions) Nylon membrane, by GE Healthcare (1 mentions) Ssc buffer, by Thermo Fisher Scientific (1 mentions)

4 protocols using dna dot blot protocol

1

Global DNA Methylation Quantification

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Genomic DNA was extracted from P21 retinae (Wizard SV genomic DNA purification Kit). Purified DNA was quantified, sonicated, denatured, and transferred to the Nylon membrane (RPN303B, GE Healthcare) according to the Cell Signaling DNA Dot Blot Protocol. 5methylcytosine (5-mC) Ab (#28692, Cell Signaling) was used to detect global DNA methylation of the samples. Methylene blue staining was used to verify equal DNA loading across samples.
Zocchi L., Mehta A., Wu S.C., Wu J., Gu Y., Wang J., Suh S., Spitale R.C, & Benavente C.A. (2020). Chromatin remodeling protein HELLS is critical for retinoblastoma tumor initiation and progression. Oncogenesis, 9(2), 25.
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2

Quantifying 5mC and 5hmC in Genomic DNA

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The genomic DNA dot-blot analysis of 5mC and 5hmC was performed following the DNA Dot Blot Protocol (Cell Signaling, #28692), as previously described.52 (link) Briefly, genomic DNA was extracted using Quick-DNA Miniprep Plus Kit (Zymo Research, D4068), and DNA concentration was measured by NanoDrop. The same amount of DNA was denatured with 10X DNA denaturing buffer (1 M NaOH and 0.1 M EDTA) and incubated at 95°C for 10 min, which was then immediately mixed with an equal volume of 20X SSC buffer, pH 7.0 (Invitrogen, 15557044) and chilled on ice. The DNA samples were diluted with a pre-determined amount and loaded on the positive-charged Nylon membrane (GE Amersham, RPN2020B) using a vacuum chamber (Manifold, SRC-96). The membrane was dried, auto-crosslinked with 1200 x100 μJ/cm2, and blocked with 5% milk/TBST for 1 h. Next, the membrane was incubated with 5mC (Cell Signaling, 28692) or 5hmC (Active Motif, 39769) antibodies, the same as the western blot analysis.
Huang X., Balmer S., Lyu C., Xiang Y., Malik V., Wang H., Zhang Y., Xie W., Hadjantonakis A.K., Zhou H, & Wang J. (2023). ZFP281 coordinates DNMT3 and TET1 for transcriptional and epigenetic control in pluripotent state transitions. bioRxiv.
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3

Genomic DNA Dot Blot Analysis of 5mC and 5hmC

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The genomic DNA dot-blot analysis of 5mC and 5hmC was performed following the DNA Dot Blot Protocol (Cell Signaling, #28692) with modifications. Briefly, genomic DNA of ESCs was extracted using Quick-DNA Miniprep Plus Kit (Zymo Research, D4068), and DNA concentration was measured by NanoDrop. Next, the same amount of DNA was denatured with 10X DNA denaturing buffer (1 M NaOH and 0.1 M EDTA) and incubated at 95°C for 10 min, which was then immediately mixed with an equal volume of 20X SSC buffer, pH 7.0 (Invitrogen, 15557044) and chilled on ice. The DNA samples were diluted with a pre-determined amount and loaded on the positive-charged Nelyon membrane (GE Amersham, RPN2020B) using a vacuum chamber (Minifold, SRC-96). The membrane was dried, auto-crosslinked with 1200 × 100 μJ/cm2, and blocked with 5% milk/TBST for 1 h. Next, the membrane was incubated with 5mC (Cell Signaling, 28692) or 5hmC (Active Motif, 39769) antibodies, the same as the Western blot analysis. Then, the membrane was stripped with the stripping buffer (Thermo Scientific, 21059) and reblotted with the dsDNA (Abcam, ab27156) antibody as the loading control.
Huang X., Bashkenova N., Hong Y., Lyu C., Guallar D., Hu Z., Malik V., Li D., Wang H., Shen X., Zhou H, & Wang J. (2022). A TET1-PSPC1-Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency. Cell reports, 39(10), 110928.
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4

Genomic DNA Dot Blot Analysis of 5mC and 5hmC

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The genomic DNA dot-blot analysis of 5mC and 5hmC was performed following the DNA Dot Blot Protocol (Cell Signaling, #28692) with modifications. Briefly, genomic DNA of ESCs was extracted using Quick-DNA Miniprep Plus Kit (Zymo Research, D4068), and DNA concentration was measured by NanoDrop. Next, the same amount of DNA was denatured with 10X DNA denaturing buffer (1 M NaOH and 0.1 M EDTA) and incubated at 95°C for 10 min, which was then immediately mixed with an equal volume of 20X SSC buffer, pH 7.0 (Invitrogen, 15557044) and chilled on ice. The DNA samples were diluted with a pre-determined amount and loaded on the positive-charged Nelyon membrane (GE Amersham, RPN2020B) using a vacuum chamber (Minifold, SRC-96). The membrane was dried, auto-crosslinked with 1200 × 100 μJ/cm2, and blocked with 5% milk/TBST for 1 h. Next, the membrane was incubated with 5mC (Cell Signaling, 28692) or 5hmC (Active Motif, 39769) antibodies, the same as the Western blot analysis. Then, the membrane was stripped with the stripping buffer (Thermo Scientific, 21059) and reblotted with the dsDNA (Abcam, ab27156) antibody as the loading control.
Huang X., Bashkenova N., Hong Y., Lyu C., Guallar D., Hu Z., Malik V., Li D., Wang H., Shen X., Zhou H, & Wang J. (2022). A TET1-PSPC1-Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency. Cell reports, 39(10), 110928.
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