Knight:Spheroplast transformation

Overview

This protocol is used to transform yeast with large DNA molecules, creating a YAC. It requires digesting away the cell wall, leaving the yeast "spheroplast" behind. The spheroplasts and the high molecular weight DNA are both very fragile - be warned, this one's fussy. If you've never done it before, we recommend reading the chapter by Green first.

Technique is still in beta testing. We are using the recombination-based cloning method of Spencer to create the YAC in vivo. This version is scaled to produce 10 plates' worth of transformants, and incorporates some modifications, mostly from Kouprina.

Materials

• DNA
  • [[Knight:Bacterial genome extraction|Source material for YAC] -- warning: requires several days
  • Linearized YAC precursor plasmids, pRML1 and pRML2 -- see Spencer

• Yeast culture
  • Strain in use is J57D (trp- ura-)
  • Log-phase overnight culture, 100 mL at ~2 x 10^7 cell/mL (OD660 of 2-4)
  • Kouprina gives the OD660 for 1/10 dilution in water as 0.14

• Stable media (can prepare in advance)
  • YPD (100 mL)
  • Sterile H2O (60 mL)
  • 1 M sorbitol (40 mL + ~5 mL)
  • SCE (40 mL)
  • 2 M DTT (200 uL)
  • Lyticase (approx. 1000 U)

• Unstable media (prepare from sterile solutions right beforehand)