X-Gal Agarose Overlay Assay From the Herskowitz Lab Protocol Solutions Required: 0.5 M Potassium Phosphate Buffer pH 7.0: (mix 61 ml of 1M K2HPO4 and 39ml of 1M KH2PO4 and add 100ml dH20) Dimethyl Formamide (DMF) 10%SDS beta-mercaptoethanol X-gal: 100mg/ml in DMF Low melt Agarose ________________________________________________________________ __________ Stock Solution: 6% DMF 0.1% SDS in 0.5M KPO4: (for 100ml mix 93 ml of phosphate buffer, 6 ml of DMF and 1 ml of 10% SDS) Notes: Use half of above if you are only overlaying up to eight large plates. Use 3.5 ml per small plates (60mmX15mm), or 7ml for large plates (100mmX15mm). ________________________________________________________________ __________ 1. Preparation of Low-Melt-Agarose: To 100ml Stock Solution add 0.5 g low melt agar and microwave in ten or fifteensecond intervals until solution becomes clear and melted. 2. Let solution cool for a minute or two while stirring then add: 2.5 ul of X-gal (100 mg/ml) for every ml of solution you are using. (you can use the unused portions of the gel for up to a week) 0.5 ul of beta mercaptoethanol (BME) for every ml of solution. (From the time you add the BME until gel solidifies it is best to work in a fume hood) 3. Apply directly onto yeast plates (3.5ml small or 7ml large plates) w/ plastic pipette or simply pour from a measured falcon tube. 4. Cover immediately to keep dark (put in drawer) and let sit for 10 minutes. 5. incubate at 30 C. Strong inducers should turn blue within 1-2 hours, weaker ones over the next 12-24 hours. 6. Colonies may be picked through the top agar for a few days later and grown on appropriate selection media.