Yale Genome Analysis Center, Yale University http://ygac.med.yale.edu/mtn/LS_transf_prot.stm The following protocols were developed by Petra Ross-Macdonald for Mike Snyder's LacZ fusion project at Yale University.

Preparing the competent cells

Transforming yeast

Equipment required

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).

Grow 50 ml culture in 2xSC-leu 1 , overnight (light innoculum, ie 20 ul of cells).

Next morning, add 40 mls YPAD, grow 2-3 hours more.

Spin down 100 ul of the culture (=1 transformation's worth) and check pellet size. Should be approx 3 ul. If much greater, use less culture for following steps 2 .

Spin down cells. 2000 r.p.m., 4 minutes. Resuspend in sterile water then spin down again.

Mix: 72 mls water, 9 mls 10xTE pH 7.5, 9 mls 1M LiAc.

Resuspend yeast in the 90 mls LiAc/TE, put back in same flask (rinsed with sterile water). Shake at 30℃ for 30 mins.

Add 600 ul beta-mercaptoethanol. Shake at 30℃ for 30 minutes.

Add 1.7ml of 10mg/ml ss carrier DNA 3 . Cells ready to use.

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?

Dispense yeast competent cells into deepwells with DNA (100 ul/well).

Capmat deepwells. Incubate at 30℃, 30 mins, on side.

Resuspend cells by vortexing.

Dispense PEG 1 into deepwell wih yeast/DNA (250 ul/well).

Pipette up and down to mix cells and PEG2 .

Capmat deepwells. Incubate at 30℃ for 15-30 mins, on side.

Heat shock at 45℃ for 15 mins.

Pellet cells at 3000 r.p.m. for 15 mins at least. Yeast form diffuse layer on bottom of well.

Tip PEG off quickly and carefully. Wipe PEG off top of box. Put caps on. Resuspend by vortexing.

To plate on solid medium:

We plate 24 transformations (2 rows of deepwell) per Nunc omniplate, using the 12 channel pipettor to put 5ul of cell suspension (in residual PEG) onto the plate, then smearing this about halfway down the plate. We get several hundred transformants from this. We get about 0-10 transformants when the DNA used should not integrate ie mTn insertions in vector.

Grow as usual for transformation 3 .

To select in liquid:

Add 580 ul of 2xSC(dropout). Put capmat on and grow with agitation4 for 3-5 days, until pellet visible.

If rough equalization of growth state is desired, pellet cells, discard medium and grow overnight with fresh medium.

Pellet cells and wash once with water. Discard water and resuspend cells by vortexing in residue.

We then add 140 ul of water, mix by pipetting, and plate 6 ul to SC-leu -ura plate. We use the rest of the suspension for filter assays and -70℃ storage.

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.

Deepwell titer dishes: Beckman 267006 ($83/24)

Capmats for same: Beckman 267005 ($22/10) (Beckman 1-800-742-2345) Alternatively try Polyfiltronics (http://www.polyfiltronics.com). Ususlly cheaper but I'm not sure what they have. Best to get caps and boxes from same source, so you can complain when they don't fit perfectly. The essential feature of these boxes is that the wells are individually formed, with gaps between so that water can get up and surround them during the heat shock.