New PDF release: Candida Species: Methods and Protocols

By Richard Calderone, Ronald Cihlar

ISBN-10: 1493930516

ISBN-13: 9781493930517

ISBN-10: 1493930524

ISBN-13: 9781493930524

This quantity presents targeted dialogue of a number of vital options that researchers use to review fungal molecular biology and pathogenesis. Written for the Methods in Molecular Biology sequence, chapters contain introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and tips about troubleshooting and keeping off recognized pitfalls.

Authoritative and sensible, Candida Species: tools and Protocols aims to make sure profitable leads to the extra examine of this important field.

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Additional resources for Candida Species: Methods and Protocols

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Rinse the sample tube with additional TRIzol and add to the homogenization vial; there should only be a small amount of air left at the top of the vial (see Note 23). 12. Shake the samples with the mechanical homogenizer for 30 s, and then chill samples on ice for 1 min or until cool to the touch (see Note 24). Repeat five times (for a total of 3 min of homogenization per sample). 13. 5 ml tubes (see Notes 25 and 26). 14. Centrifuge at 12,000 × g for 10 min at 4 °C (see Note 27). 15. 5 ml tube (see Note 28).

Start 5 ml LB-amp primary liquid culture of single colony (for plasmid preparation). – Incubate secondary culture diluting from the primary culture 1:100 in LB-Amp media overnight or for a maximum of 16–18 h at 37 °C with 220 rpm shaking. Store the E. coli cells at −80 °C (see Note 1). Plasmid preparation (E. ). – Plasmid DNA is purified from the stored E. coli cells using a standard kit. 8 % agarose gel and is quantified spectrophotometrically. The purified plasmid is digested with SmaI in the following way: Remove 3 μl of each plasmid for checking the uncut DNA.

13. Harvest the 24-h culture by transferring it to a 50 ml conical tube; centrifuge at 3220 × g for 6 min. 14. Discard the supernatant. Wash the pellet with 20 ml sterile PBS. Centrifuge at 3220 × g for 6 min. 15. Repeat step 14 once. 16. Discard the supernatant. Resuspend the pellet in 3 ml sterile PBS. 17. Dilute 5 μl of cell suspension in 1 ml PBS (1:200 dilution). Count the cells and prepare 5 ml of cell suspension at a concentration of 5 × 108 cells/ml (in sterile PBS). W. Gunsalus and Carol A.

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

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