By Richard Calderone, Ronald Cihlar
This quantity offers unique dialogue of various vital strategies that researchers use to review fungal molecular biology and pathogenesis. Written for the Methods in Molecular Biology sequence, chapters comprise introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, easily reproducible laboratory protocols, and tips about troubleshooting and averting identified pitfalls.
Authoritative and functional, Candida Species: equipment and Protocols aims to make sure winning ends up in the additional learn of this important field.
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Additional resources for Candida Species: Methods and Protocols
Proteinase K (Thermo Scientific Cat. No. EO0492). 11. Phenol:chloroform:isoamyl alcohol (Tris saturated) (25:24:1) (Sigma Cat. No. P2069) (see Note 4). 12. Ethanol. 13. 4 M LiCl. 14. Agarose. 15. PCR reagents. 16. Autoclaved distilled water. 2 Buffers 1. 4), 10 mM Dithiothreitol (Sigma Cat. No. D0632). Make the buffer just before use. 2. 2 M Sorbitol. 3. 2 M Sorbitol, 1 mM MgCl2. 4. 10 mM Na2HPO4. 5. 5). 6. 5). 7. 1 % Candida ChIP Assay 47 Na-deoxycholate. Mix well to dissolve detergent, filter sterilize, and store at 4 °C.
The basic principle behind this technique involves the use of a small, easily diffusible and reversible cross-linking agent (like formaldehyde) for capturing in vivo protein–DNA interactions. ), Candida Species: Methods and Protocols, Methods in Molecular Biology, vol. 1007/978-1-4939-3052-4_4, © Springer Science+Business Media New York 2016 43 44 Sreyoshi Mitra et al. fragments which are then immunoprecipitated (IP) with antibodies specific to the protein of interest. During immunoprecipitation, the DNA sequences associated with the protein are preferentially pulled down from the sheared chromatin, thereby giving a realistic approximation of the genomic regions associated with the protein.
2. Oligo(dT)20 (50 μM). 3. dNTP mix: 10 mM each dATP, dGTP, dCTP, and dTTP at neutral pH. 4. RNase inhibitor. 1 Methods Animal Models 1. Upon arrival, house mice three to four per cage in autoclaved, enclosed, filter-top cages under pathogen-free conditions with sterile food and water. Let the mice acclimate for 7 days prior to inoculation. Transcriptional Profiling in the Host 21 2. Four days prior to inoculation, streak strains from glycerol stocks onto YPD agar plates. Incubate at 25 °C for 3 days or 30 °C for 4 days.