Mutagenizing a yeast gene with a mini-transposon
First, clone gene into vector pHSS6. Then:
- Day 1: Transform plasmid into bacteria expressing transposase.
- Day 2: Mate transformants to bacteria carrying mTn (Mating means mixing the cells and plating with selection).
- Day 3: Scrape off cells and mate to bacteria carrying resolvase.
- Day 4: Scrape off cells, make a miniprep and transform into E. coli.
- Day 5: Scrape off transformants, make a miniprep, cut with NotI.
- Day 6: Transform digest into yeast.
It's just that simple.