COS7 cell transfection experiments were performed with Lipofectamine 2000 ( Life Technologies Carlsbad, CA) . Primary cultured mouse cortical neurons were prepared essentially as described44 (link). Briefly, cerebral cortices of ICR mouse embryos at E14.0 were microdissected and treated with DNaseI and Papain (Nacalai Tesque, Kyoto, Japan). Then, plasmids were transfected into the primary cortical cells by NEPA21 (NEPAGENE, Chiba, Japan). SDS-PAGE (10% gel) and western blotting were performed as described45 (link). For all western blots, uncropped blot data were provided in Supplementary Information. Immunofluorescence analyses were carried out as described46 (link). Alexa Fluor 488- or 568-labeled IgG (Life Technologies) was used as a secondary antibody. Fluorescent images were captured with FV-1000 confocal laser microscope (Olympus, Tokyo, Japan).
Dnase 1
Manufactured by Nacalai Tesque
Sourced in Japan
DNase I is an enzyme that catalyzes the degradation of DNA. It is commonly used in molecular biology and biochemistry applications to remove DNA from samples.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Takara Bio (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Nacalai Tesque (2 mentions)
Papain, by Nacalai Tesque (1 mentions)
Alexa fluor 488 or 568 labeled igg, by Thermo Fisher Scientific (1 mentions)
Fv1000 confocal laser microscope, by Olympus (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Nepa21, by Nepa Gene (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Eagle s minimal essential medium, by Nissui Pharmaceutical (1 mentions)
Hydrogen peroxide, by Fujifilm (1 mentions)
Skim milk, by Fujifilm (1 mentions)
Trichloroacetic acid, by Fujifilm (1 mentions)
Protein a sepharose fast flow column, by GE Healthcare (1 mentions)
Mouse monoclonal anti tubulin antibody, by Merck Group (1 mentions)
Rnase a, by Nacalai Tesque (1 mentions)
O phenylenediamine, by Fujifilm (1 mentions)
Proteinase k, by Merck Group (1 mentions)
0.2 μm pore size filter, by Merck Group (1 mentions)
Proteinase k, by Fujifilm (1 mentions)
Rnase a, by Merck Group (1 mentions)
4 protocols using dnase 1
1
COS7 Transfection and Cortical Neuron Experiments
2
Isolation of Bacterial Genomic DNA from L929 Cells
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The bacterial cells were inoculated to L929 cells and incubated in Eagle’s minimal essential medium (Nissui, Japan) containing 1% fetal bovine serum and 2 mM L-glutamine at 34°C for 7–10 days as previously described (Fujita, 2008 ). After centrifugation at 800 rpm for 5 min to remove L929 cell debris, the supernatants were centrifuged at 14,000 rpm for 10 min to recover bacterial cells. The obtained pellets were suspended in phosphate-buffered saline (PBS, pH 7.2). The cell suspensions were treated with DNase I (Takara Bio, Japan; 2,000 U/ml at a final concentration) for 1 h at 37°C to digest cell-free DNA. DNase I was inactivated by incubation at 65°C for 5 min, and EDTA (Nacalai Tesque, Japan) was added to the solutions at a final concentration of 2 mM. Bacterial genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Japan) according to the manufacturer’s instructions.
3
Radioimmunoprecipitation Assay (RIPA) Protocol
4
Prophage Induction and DNA Isolation
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Bacterial cells were grown overnight to the stationary phase at 37°C in lysogeny broth (LB) medium. For prophage induction, cells were grown to the late log phase (0.7–0.9 OD600), and MMC was added to the culture to a final concentration of 1 μg/ml. After a 3-hr incubation, aliquots of the culture were isolated, and the cells were collected by centrifugation. Total cellular DNA was extracted from the cells using the alkaline-boiling method and used for PCR analyses. Phage particles were isolated from the culture supernatant after a 3-hr incubation with MMC. The culture was first treated with chloroform, and bacterial cell debris was removed by centrifugation. The supernatant was filtered through a 0.2-μm-pore-size filter (Millipore) and incubated with DNase I (final concentration: 400 U/ml, TaKaRa) and RNase A (50 μg/ml, Sigma) at 37°C for 1 hr. After inactivating DNase I by incubation at 75°C for 10 min and adding EDTA (5 mM, Nacalai Tesque), the sample was treated with proteinase K (100 μg/ml; Wako) and used as packaged phage DNA. Total cellular DNA and packaged phage DNA from MMC-untreated cultures were prepared with the same protocol. The primers used in these analyses are listed in S6 Table .
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