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Large scale yeast transformation

Large-scale yeast transformation

The following protocols were developed by Petra Ross-Macdonald for Mike Snyder's LacZ fusion project at Yale University.

Yeast Cell Preparation

This protocol merges a typical LiAc protocol with the reducing agent component of the One-step method. It gives>103/ ug efficiency for our strain. Recipe is for 800 transformations (90 mls cells).

  1. We use selection because we are maintaining a plasmid. Presumably the main thing is that cells not be stationary.
  2. If you are going to plate transformations, a larger pellet could be used - up to 10 ul?
  3. Boehringer 146-7-140. We omitted this for months. It didn't seem to affect transformation efficiency at all, but maybe it affects non-homologous integration?

Large Scale Transformation

To plate on solid medium:

To select in liquid:

  1. PEG: 400 mls 50% PEG, 50 mls 10x TE pH 7.5, 50 mls 1M LiAc pH7.5. We use relatively fresh mix, but this could be superstition.
  2. I guess you could invert, but the capmats don't seal brilliantly. Could use plate sealers (e.g. Corning Costar from VWR 29442-310, $24/100)
  3. Agar in omniplates seems to dry out quicker, so make sure incubator is humid.
  4. I had the Yale machine shop make metal holders that hold 3 deepwell boxes each. I attached these to the flat disc of a vertical rotating wheel (Glas-Col rugged rotator, from Fisher). The boxes thus get rotated, pretty much like tubes in a roller drum. Shaking vertically in an orbital would probably be okay too.

Equipment required

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