Mutagenizing a yeast gene with mTn-lacZ/LEU2【Yale University】

Screening for in-frame lacZ fusions in yeast

1. Transformant colonies are patched to SC -leu.
2. Cells are replica plated to a SC -leu plate and a SC-leu plate on which a sterile disc of Whatman 1A filter paper has been placed, and grown overnight at 30^oC. Other media or growth conditions can be substituted as desired. For ade2 strains, any test media should contain 80 mg/l of adenine, as the red pigment can obscure the X-gal result.
3. Filters are lifted from the plates and placed in the lid of a 9-cm glass petri dish. This lid is then placed inside a closed 15-cm glass petri dish containing chloroform, for 10 to 30 minutes. The minimum exposure time necessary for a particular yeast strain can be determined empirically.
4. Filters are placed colony-side up onto X-Gal plates (120 ug/ml 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside, 0.1 M NaPO4 [pH 7] and 1 mM MgSO4 in 1.6% agar) and incubated at 30^oC for up to 2 days.
5. Transformants carrying productive lacZ fusions are recovered from the regrown SC-leu plate . It is advisable to subsequently maintain selection for LEU2 wherever possible, as some mutations are deleterious even in the heterozygous state.

Bacterial strains used (included in kit):

Vector used:

The accession for pHSS6 is M84115

Antibiotics used:

When only a few plates of each type are used, it's convenient to chop an LB plate up with a sterile toothpick, put the bits in a sterile flask, and melt the agar by microwave. Add appropriate amounts of antibiotic and repour plates.