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
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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).
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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.