X-Gal overlay assay for Yeast

X-Gal Agarose Overlay Assay 

From the Herskowitz Lab Protocol

Solutions Required:

.5 M Potassium Phosphate Buffer pH 7.0: (mix 61ml of 1M K2HPO4 and 39ml of 1M KH2PO4 and add 100ml dH20)

Dimethyl Formamide (DMF)

10%SDS:  10g in 100ml H2

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 93ml of phosphate buffer, 6ml 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 fifteen-second intervals until solution becomes clear and over 65C.

2. Let solution cool for a minute or two then add:

10ul of Xgal (100mg/ml) for every ml of solution you are using. (you can use the unused portions of the gel for up to a week)

0.5ul 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 with foil (to keep dark) and let sit for 10 minutes.

5. Wrap in parafilm and put at 30C. 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 if appropriate selection media.

 

Notes:  We find that this method is less sensitive than the "standard" lacZ filter lifts and can be useful if  your bait in the yeast two hybrid assay has  weak self activation.