| | Yeast Nitrogen Base | pH adjustment1 | 48 hrs observation | 60 hrs observation | 72 hrs observation |
|---|
| | | |
Gal4
|
Ubx
|
Pro-2
|
Gal4
|
Ubx
|
Pro-2
|
Gal4
|
Ubx
|
Pro-2
|
1 | Sigma | + | ++ | - | - | +++ | + | - | ++++ | ++ | ++ |
2 | Clontech | + | - | - | - | ++ | - | - | +++ | ++ | ++ |
3 | Difco | + | ++ | - | - | +++ | + | - | +++ | ++ | ++ |
4 | Sigma | - | +++ | - | - | ++++ | +++ | ++ | ++++ | +++ | ++ |
5 | Clontech | - | ++ | + | - | +++ | ++ | + | +++ | ++ | ++ |
6 | Difco | - | - | - | - | ++ | - | - | ++++ | +++ | - |
- We examined six media conditions, defined by yeast nitrogen base supplier and pH adjustment post-autoclave. Difco media can yield false negative data (condition 6) and the other media preparations can yield false positives (conditions 1-5) compared to other transcription activation assay methods [8]. “pH” refers to the pH of the 0.7 M potassium phosphate buffer (KP); “Gal4” stands for pLexA-Gal4, the positive control plasmid expressing the DNA binding domain of LexA fused to the Gal4 transcription activation domain (Clontech). “Ubx” represents the pLexA-Ubx plasmid, “Pro-2” is the negative control, in which proline mutations disrupt transcription activation by the LexA-Ubx hybrid [8]. The blue shade of colonies is reported here as symbols: “++++” for dark blue, “+++” for medium blue, “++” for light blue, “+” for pale blue, and “–” for white colonies. See Additional File 2 for color standards. The pH after autoclaving but before addition of KP was 4.67, 5.11, and 4.31 for Sigma, Clontech, and Difco media, respectively. After addition of KP buffer post-autoclaving, the pH of all media was 6.7 ± 0.1. For conditions 1-3, the pH was adjusted to 7.0. Therefore pH differences are not the source of the supplier-based differences in results.
