An open access editable version of this protocol is available through Nature Protocol Exchange at: http://dx.doi.org/10.1038/protex.2013.082. A description of the components of the holidic medium as well as supplier order numbers for all components can be found in Table 1. Preparation of the medium is performed in two stages. In the first, sucrose, agar, amino acids with low solubility (L-isoleucine, L-leucine and L-tyrosine) as well as stock solutions of buffer, metal ions and cholesterol are combined in a 1 liter autoclavable bottle with a magnetic stirrer and milliQ water up to 1 liter, minus the volume of solutions to be added after autoclaving. Following autoclaving at 120 °C for 15 min, the solution is allowed to cool at room temperature with stirring to ~65 °C. Stock solutions for the amino acids (Table 2), vitamins, nucleosides, choline, inositol and preservatives are then added as are the drugs mifepristone (Sigma) or rapamycin (LC Laboratories), where appropriate. With constant stirring, sterile tubing is used to dispense the solution into sterile vials. These are covered, allowed to cool for 90 min at room temperature and then stored at 4 °C until use. Feedback from other users of the medium have highlighted that this method of preparation may result in media that does not set. This varies with the autoclave used and can be resolved by adding the sterilized buffer base after autoclaving, indicating that it is caused by acid hydrolysis of the agar during autoclaving.
Stock solutions are prepared in milliQ water, except for the cholesterol stock, which is prepared in absolute ethanol. The cholesterol stock, buffer stock, amino acid solutions and stock containing nucleosides, choline and inositol are stored at 4 °C, while the FeSO4, vitamin and folic acid stocks are stored at –20 °C. Before freezing of these latter stocks, we would typically make 1 liter and make aliquots of smaller volumes so that once thawed, they could be used quickly and without re-freezing. Before storing, amino acid stocks are pH adjusted to 4.5 using HCl. All aqueous solutions were filter sterilized by passing through a 0.22 μm syringe-fitted filter. Note also, that cholesterol precipitates out of solution during storage at 4°C, but it easily re-dissolves with stirring at room temperature. The amino acid ratio shown in Table 1 refers to HUNTaa. The proportions and amounts of the amino acid stock solutions as well as their value in terms of biologically available nitrogen can be found in Table 2.