This protocol describes standard methods for constructing a yeast single gene expression clone (or Transcription Unit) using the yeast pathway kit.

The first step is to prepare the pYPK0 or pYPKpw vector. The pYPKpw vector is easier to use since it has a deletion theoretically preventing partial clones.

Use a standard miniprep kit if available, but the [low cost protocol](super snik) yields sufficiently pure DNA for this purpose.

pYPKpw can be linearized with blunt enzymes: ZraI, FspAI EcoRV/Eco32I

The digestion below is enough for ten ligations.

• 6 µL water
• 1 µL pYPKpw miniprep
• 1 µL 10X Buffer EcoRV
• 1 µL EcoRV (5U/µL)
• 1 µL SAP, FastAP or some other alkaline phosphatase

Inactivate ZraI, EcoRV, Eco32I at 80°C for 20 min. It is practical to do the above digestion/inactivation in a PCR thermal cycler in available. Put the digestion on ice immediately after the digestion has finished.

The digested vector can be saved at -20°C at this point.