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[DESCRIPTION]
This recipe is from: <br /> <span class="cit-auth cit-auth-type-author">Alexander WG</span> <span class="cit-sep cit-sep-separator">, </span> <span class="cit-auth cit-auth-type-author">Doering DT</span> <span class="cit-sep cit-sep-separator">,</span> <span class="cit-sep cit-sep-last-separator">and</span> <span class="cit-auth cit-auth-type-author"> Hittinger CT (2014) <a href="http://www.genetics.org/content/198/3/859.full" target="_blank">High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces</a>. Genetics <span class="cit-vol">198<span class="cit-sep cit-sep-after-article-vol">:</span></span><span class="cit-pages"><span class="cit-first-page">859</span><span class="cit-sep">-</span><span class="cit-last-page">866; </span></span><span class="cit-doi"><span class="cit-sep cit-sep-before-article-doi">doi:</span>10.1534/genetics.114.170118<br /><br />See the <a href="https://www.protocols.io/view/HERP-Haploid-Engineering-and-Replacement-Protocol-drq55v" target="_blank">full protocol</a> for step-by-step YPGly+ AF preparation instructions.<br /></span></span>

[GUIDELINES]
This recipe is from: <br /> <span class="cit-auth cit-auth-type-author">Alexander WG</span> <span class="cit-sep cit-sep-separator">, </span> <span class="cit-auth cit-auth-type-author">Doering DT</span> <span class="cit-sep cit-sep-separator">,</span> <span class="cit-sep cit-sep-last-separator">and</span> <span class="cit-auth cit-auth-type-author"> Hittinger CT (2014) <a href="http://www.genetics.org/content/198/3/859.full" target="_blank">High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces</a>. Genetics <span class="cit-vol">198<span class="cit-sep cit-sep-after-article-vol">:</span></span><span class="cit-pages"><span class="cit-first-page">859</span><span class="cit-sep">-</span><span class="cit-last-page">866; </span></span><span class="cit-doi"><span class="cit-sep cit-sep-before-article-doi">doi:</span>10.1534/genetics.114.170118<br /><br />See the <a href="https://www.protocols.io/view/HERP-Haploid-Engineering-and-Replacement-Protocol-drq55v" target="_blank">full protocol</a> for step-by-step YPGly+ AF preparation instructions.<br />------------------------<br />1) Add the following components to a 2‐L Erlenmeyer flask:<br />‐10 g yeast extract<br />‐20 g peptone<br />‐5 g sulfanilamide<br />‐50 mg hypoxanthine<br />‐18 g agar<br />‐900 mL ddH2O<br /><br />Mix to dissolve as much as possible (agar and sulfanilamide won't dissolve until heated).<br /><br />2) Autoclave for no more than 20 minutes on a liquid cycle.<br /><br />3) Once autoclaved, cool to 50° in a water bath, then add the following and mix:<br />‐5 g thymidine<br />‐200 mg methotrexate<br />‐100 mL 50% (v/v) glycerol, sterilized<br />(<strong>NOTA BENE</strong>: the standard operating procedure for adding compounds after autoclaving is to dissolve them in a<br />solvent, filter, then add to the media; this generally is difficult or impossible for methotrexate and thymidine due to<br />the amount required. For the last two years, I've been adding the solid chemicals directly to the cooled media, and<br />I've never had contamination. I suspect that the extreme conditions prevent microbial growth. Also, both<br />methotrexate and thymidine are sensitive to heat, so take care to not add them early.)<br /><br />4) Pour ~20 mL into plastic petri dishes and allow to set. You have now made YPGly +AF media<br /></span></span>

[STEPS]
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