Download Candida Species: Methods and Protocols by Richard Calderone, Ronald Cihlar PDF

By Richard Calderone, Ronald Cihlar

This quantity offers designated dialogue of numerous vital thoughts that researchers use to review fungal molecular biology and pathogenesis. Written for the Methods in Molecular Biology sequence, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, easily reproducible laboratory protocols, and pointers on troubleshooting and fending off identified pitfalls.

Authoritative and useful, Candida Species: equipment and Protocols aims to make sure winning ends up in the additional examine of this very important field.

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Plasmids required: Plasmid #1: “pCW778” (markers: HIS3, URA3), deletes the stuffer URA3 marker upon SmaI digest. Plasmid #2: “pYL040” (markers: URA3, HIS3), deletes the stuffer HIS3 marker upon SmaI digest. Figure 2a, b (Plasmid Maps). The plasmid pYL40 contains the fragment of STE11 encoding Ste11p lacking its SAM domain (aa 105–717) at the SalI site 34 Jaideep Mallick et al. SacI PmeI AscI a F MscI GAL1 F NotI SpeI BamHI SmaI GAL1 NheI SmaI AatII HIS3 Stuffer ampR SacI PmeI AscI b NotI SpeI BamHI SmaI URA3 Stuffer R ampR R STE50ΔSAM STE11ΔSAMmyc pCW778 pYL40 tcyc1/ loxP URA3 tcyc1/ loxP kanMX SmaI AatII loxP XhoI PacI kanMX HIS3 MscI NheI XhoI loxP PacI c bait**/prey** Ste50/Ste11ΔSAM Stuffer in vivo recombination bait**/prey** Ste50/Ste11ΔSAM Fig.

6 Input (I) DNA Purification 1. 0) and 5 μl RNase A (10 mg/ml). Incubate at 37 °C for 1 h. 2. Add 25 μl Proteinase K (20 mg/ml). Incubate at 37 °C for 4 h. 3. 3 ml of phenol:chloroform:isoamyl alcohol and vortex for 10 s. 4. 5 ml Eppendorf tubes. 5. Add 1 ml of 100 % ethanol per tube and vortex. Precipitate DNA at room temperature for ≥15 min. 6. Spin down the precipitate in a microcentrifuge at room temperature, 19200 × g for 20 min. 52 Sreyoshi Mitra et al. 7. Wash DNA pellet with 1 ml of 70 % ethanol.

Invitrogen 19. White SJ, Rosenbach A, Lephart P, Nguyen D, Benjamin A, Tzipori S, Whiteway M, Mecsas J, Kumamoto CA (2007) Selfregulation of Candida albicans population size during GI colonization. PLoS Pathog 3(12):e184. 1371/journal. 0030184 20. Pierce JV, Kumamoto CA (2012) Variation in Candida albicans EFG1 expression enables host-dependent changes in colonizing fungal populations. MBio 3(4):e00117–e00112. 00117-12 21. Kumamoto CA (2011) Inflammation and gastrointestinal Candida colonization.

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