By Richard Calderone, Ronald Cihlar
This quantity presents exact dialogue of quite a few very important concepts that researchers use to review fungal molecular biology and pathogenesis. Written for the Methods in Molecular Biology sequence, chapters comprise introductions to their respective issues, lists of the required fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and tips about troubleshooting and heading off recognized pitfalls.
Authoritative and sensible, Candida Species: equipment and Protocols aims to make sure profitable ends up in the additional learn of this important field.
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Additional info for Candida Species: Methods and Protocols
33. Add water to the reaction first, then oligo(dT), and RNase inhibitor; add RNA last. Use oligo(dT) instead of random primers because this selects for eukaryotic mRNAs, and eliminates much of the contamination from bacterial RNAs. 34. Dilute cDNA 1:10 or 1:20 in nuclease-free water; do not use undiluted cDNA, as it typically contains inhibitors that will interfere with efficient qPCR amplification. If aliquots are prepared in 8-well strip tubes, a multichannel pipette can be used to add cDNA to qPCR reaction tubes.
Incubate at 30 °C overnight (see Note 6) (Fig. 3a). 75 NaCl) Fig. 3 Schematic analysis of protein-protein interaction: (a) Candidate proteins are cloned in both bait and prey configurations through in vivo recombination in two isogenic, osmo-sensitive yeast strains (ste50Δ ssk2Δ ssk22Δ of opposite mating types (yCW1476 and yCW1477)). Stuffer marker negative strains are selected to ensure that there is a candidate insert. The recombinants of bait and prey were crossed and diploid cells containing both bait and prey were selected to assay whether bait and prey interact by testing their ability to activate the HOG pathway as scored for growth on hyperosmotic media.
38 Jaideep Mallick et al. 100–150 μl of S. cerevisiae cells in the transforming mix (see Note 4). The IVR work is as follows: yeast cells are transformed simultaneously with the cut vector and the PCR-amplified product of the insert one wants to put into the vector. 7 Day 6 Day 7 Day 8 – Incubate the mixture for 30 min at 30 °C (in an incubator, no water bath required). – Heat shock the suspended cells at 42 °C for 20 min. – Wait for 2 min for the cells to cool to room temperature. Centrifuge the mixture at 4000 × g for 1 min at room temperature, remove the supernatant, and resuspend the cells in sterile distilled water (optional).
Candida Species: Methods and Protocols by Richard Calderone, Ronald Cihlar