TRAFO Efficiency: up to 22 million transformanst/ µg of plasmid DNA
HIGH EFFICIENCY TRANSFORMATION

Please cite:
Agatep, R., R.D. Kirkpatrick, D.L. Parchaliuk, R.A. Woods, and R.D. Gietz (1998) Transformation of Saccharomyces cerevisiae by the lithium acetate/single-stranded carrier DNA/polyethylene glycol (LiAc/ss-DNA/PEG) protocol. Technical Tips Online (http://tto.trends.com) for this method.

All Solutions for the LiAc/SS-DNA/PEG TRAFO Protocol are listed in the TRAFO Solutions Page.

1. Inoculate 2-5 mls of liquid YPAD or 10 ml SC and incubate with shaking overnight at 30^oC.
2. Count o/n culture and inoculate 50 mls of warm YPAD to a cell density of 5 x 106/ml culture.

Note:

i) Dilute overnight YPAD or SC cultures 10-1 or more in water.

ii) Carefully place 10 µl of the cell suspension between the cover slip and the baseof haemocytometer. Let the cells settle onto the haemocytometer grid for a fewminutes. The grid area is typically 1 square millimeter, divided into 25 equal-sized squares, and the volume measured is 10-4 ml.

ii) Count the number of cells in 5 diagonal squares

iv) Calculate the cell titer as follows: cells counted x 5 x dilution factor x 1/volume measured by the 25 squares of the haemocytometer. 239 cells x 5 x 10 (dilution factor) x 1/10-4ml =1.2 x 108 cells/ml.

v) Saccharomyces cerevisiae divides by budding from a mother cell. Count budded cells as a single cells. Count cells with equal bud sizes as two cells there is evidence of additional buds forming on either cell. Some strains form clumps of cells which reduce plating efficiency. A single clump of cells will only give rise to one colony on a plate, which may complicate further analysis.

vi) You may also use OD600 to determine cell titer, however the relationship between cell number and OD is strain specific.

3. Incubate the culture at 30^oC on a shaker at 200 rpm until it's equivalent to 2 x 107 cells/ml. This will take 3 to 5 hours. This culture will give sufficient cells for 10 transformations.

Note:

i) It is important to allow the cells to complete at least two divisions.
ii) Transformation efficiency (transformants/ µg plasmid/108 cells) remains constant for 3 to 4 celldivisions.

4. Harvest the culture in a sterile 50 ml centrifuge tube at 3000 x g (5000 rpm) for 5 min.
5. Pour off the medium, resuspend the cells in 25 ml of sterile water and centrifuge again.
6. Pour off the water, resuspend the cells in 1.0 ml 100 mM LiAc and transfer the suspension to a 1.5 ml microfuge tube.
7. Pellet the cells at top speed for 15 sec and remove the LiAc with a micropipette.
8. Resuspend the cells to a final volume of 500 µl (2 x 109 cells/ml) --about 400µl of 100 mM LiAc--

Note:

If the cell titer of the culture is greater than 2 x 107/ cells ml the volume of the LiAc should be increased to maintain the titer of this suspension at 2 x 109 cells/ml. If the titer of the culture is less than 2 x 107/ cells ml then decrease the amount of LiAc.

9. Boil a 1.0 ml sample of SS-DNA for 5 min. and quickly chill in ice water.

**** It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your own freezer box and boil after 3-4 freeze-thaws. But keep on ice when out.****

10. Vortex the cell suspension and pipette 50 µl samples into labelled µfuge tubes. Pellet the cells and remove the LiAc with a micropipette.
11. The basic "transformation mix" consists of:

240 µl PEG (50% w/v)
36 µl 1.0 M. LiAc
50 µl SS-DNA (2.0 mg/ml)
X µl Plasmid DNA (0.1 - 10 µg)
34-X µl Sterile ddH2O
360 µl TOTAL

Carefully add these ingredients in the order listed.

Note:

The order is important here! The PEG should go in first, which shields the cells from the detrimental effects of the high concentration of LiAc.

One can also premix the ingredients except for the plasmid DNA then add 355µl of TRAFO mix ontop of the cell pellet. Then add the 5 µl of plasmid DNA and mix. Take care to deliver the correct volume as the Trafo mix is viscous.

12. Vortex each tube vigorously until the cell pellet has been completely mixed. Usually takes about 1 min.
13. Incubate at 30^oC for 30 min.
14. Heat shock in a water bath at 42^oC for 30 min.

Note:

The optimum time can vary for different yeast strains. Please test this if you need high efficiency from your transformations.

15. Microfuge at 6-8000 rpm for 15 sec and remove the transformation mix with a micropipette.
16. Pipette 1.0 ml of sterile water into each tube and resuspend the pellet by pipetting it up and down gently.

Note:

We like to be a gentle as possible at this step if high efficiency is important. Excessive washing washes away transformants!!!!

17. Plate from 2 to 200 µl of the transformation mix onto SC-minus plates. If plating less than 200 µl deliver into a pool of not more than a final volume of 200 µl of sterile water on the plate.

Note:

When spreading yeast inoculum onto the plate gently distribute the fluid completely with a sterile glass rod with a minimum of strokes. Allow the fluid to be taken up by the plate prior to incubation.

18. Incubate the SC minus plates for 2 - 4 days to recover transformants.

Note:

Two plasmids, such as an expression plasmid and a library plasmid pool, can be co-transformed into a single cell by including both plasmids in the same transformation reaction. The efficiency is reduced, however, because only about 30-40 % of all transformed cells take up more than one plasmid molecule. An alternative approach, which has a higher efficiency is to transform in the expression plasmid first, using this or the Quick and Easy protocol, and then use the protocol found on the 2 Hybrid System TRAFO Page to transform in the library plasmid pool.