Thymidine dt

Manufactured by Merck Group

Thymidine (dT) is a nucleoside used in molecular biology and biochemistry applications. It is a building block of DNA and serves as a precursor for the synthesis of thymidine triphosphate (dTTP), which is incorporated into DNA during replication.

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Lab products found in correlation

Diaminopimelic acid dap, by Merck Group (2 mentions) Spectinomycin spc, by Merck Group (1 mentions) Emjh medium, by Bio-Rad (1 mentions) Kanamycin km, by Merck Group (1 mentions) Cisplatin, by STEMCELL (1 mentions) Doxorubicin, by Merck Group (1 mentions) Ham s f 12k medium, by Thermo Fisher Scientific (1 mentions) Nu7441, by STEMCELL (1 mentions) Penicillin streptomycin, by Corning (1 mentions) Hydroxyurea hu, by Merck Group (1 mentions) Dulbecco modified eagle medium (dmem), by Corning (1 mentions) Cisplatin, by Merck Group (1 mentions)

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Thymidine

4 protocols using thymidine dt

Culturing Leptospira Strains for Genetic Manipulation

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Saprophytic L. biflexa serovar Patoc strain Patoc1 and low-passage pathogen L. interrogans serovar Copenhageni strain Fiocruz L1-130 were cultured in EMJH medium (Difco, BD, Franklin Lakes, NJ, United States) supplemented with 10% (vol/vol) Leptospira Enrichment EMJH (Difco) (Turner, 1970 (link)). Solid media were prepared by supplementing with 1.2% noble agar (Difco). Where necessary, spectinomycin was added at a concentration of 40 μg/ml. E. coli auxotrophic strains π1 and β2163 (Demarre et al., 2005 (link); Picardeau, 2008 (link)) were used for general cloning and as conjugation donor cells, respectively, and were grown in Luria–Bertani (LB, Difco) medium. For E. coli strain π1, thymidine (dT, 0.3 mM, Sigma) was added to the medium, and for strain β2163, diaminopimelic acid (DAP, 0.3 mM, Sigma) was added.

Fernandes L.G, & Nascimento A.L. (2022). A Novel Breakthrough in Leptospira spp. Mutagenesis: Knockout by Combination of CRISPR/Cas9 and Non-homologous End-Joining Systems. Frontiers in Microbiology, 13, 915382.

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Leptospira and E. coli Cultivation Protocol

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Pathogenic L. interrogans serovar Copenhageni strain FIOCRUZ L1-130 and a recently isolated pathogenic non-interrogans Leptospira strain LGVF02 from soil samples (Nathan Stone, Northern Arizona University, manuscript in preparation) were grown in liquid HAN media^{23 (link)} at 29 °C. Saprophytic L. biflexa serovar Patoc strain Patoc1 was cultured in EMJH medium (Difco, BD, Franklin Lakes, NJ). Solid media were prepared by supplementing with 1.2% noble agar (Difco). Where necessary, spectinomycin was added at 40 µg/mL. E. coli strains π1 and β2163^{24 (link)} were used for general cloning and as conjugation donor cells, respectively, and were grown in Luria–Bertani (LB, Difco) media. For E. coli strains π1, thymidine (dT, 0.3 mM, Sigma) was added to the media and for strain β2163, diaminopimelic acid (DAP, 0.3 mM, Sigma) was added.

Fernandes L.G., Hornsby R.L., Nascimento A.L, & Nally J.E. (2021). Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C. Scientific Reports, 11, 1768.

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Propagation and Maintenance of Leptospira

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L. biflexa sevorar Patoc strain Patoc (Paris) and L. interrogans serovar Manilae strain L495 were used in this study as described earlier [17] (link). Bacteria were grown in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (Bio-Rad) at 28°C without agitation. EMJH agar plates were obtained by solidification of EMJH medium with 1.2% noble agar (Difco). Leptospires were counted using a Petroff-Hauser chamber. Antibiotics were used in vitro at the following concentrations: 100 µg/mL ampicillin (Amp, MP Biomedical); 25 µg/ml kanamycin (Km, Sigma-Aldrich); 50 µg/ml spectinomycin (Spc, Sigma-Aldrich). Thymidine (dT, Sigma-Aldrich) and diaminopimelate (DAP, Sigma-Aldrich) were added when necessary at the final concentration of 0.3 mM.

Ratet G., Veyrier F.J., Fanton d'Andon M., Kammerscheit X., Nicola M.A., Picardeau M., Boneca I.G, & Werts C. (2014). Live Imaging of Bioluminescent Leptospira interrogans in Mice Reveals Renal Colonization as a Stealth Escape from the Blood Defenses and Antibiotics. PLoS Neglected Tropical Diseases, 8(12), e3359.

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Cell Culture and Synchronization Assay

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T98G, MCF7, and U2OS cells were obtained from ATCC and regularely tested for mycoplasma by PCR. They were cultured in DMEM (Cellgro, 10–017-CV), 10% fetal bovine serum, and 1% penicillin/streptomycin (Cellgro, 30–002-Cl). PC3 cells were cultured in Ham’s F-12K medium (Gibco, 21127022), 10% fetal bovine serum and 1% penicillin/streptomycin (Cellgro, 30–002-Cl). All cultures were maintained in 5% CO2 at 37°C. Unless indicated otherwise, cells were treated with 25 ug/mL cisplatin (Sigma, 15663–27-1) or 15 ug/mL doxorubicin (Sigma, 25316–40-9) overnight. For DNA-PK inhibition, cells were treated with 10 nM NU7441 (STEMCELL, A8315) for 30 minutes before being treated with cisplatin. Cells were treated with 2.5 uM thymidine (dT, Sigma) for 24 hours, then released into media with 100 ng/mL nocodazole (ND, Sigma) for G2/M synchronization. Cells were treated with 1 uM hydroxyurea (HU, Sigma) for 24 hours to prevent S phase progression.

Sugiyama T., New J.H, & Kowalczykowski S.C. (1998). DNA annealing by Rad52 Protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA. Proceedings of the National Academy of Sciences of the United States of America, 95(11), 6049-6054.

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