LiAc/SS-DNA/PEG TRANSFORMATION

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.

This protocol was drafted by Vinny Guacci circa 2013 with some modifications by Thomas Eng.

1. Inoculate 2-5 mls of liquid YPAD and incubate with shaking overnight at 30^oC on a roller drum (speed: ~8).

2. The next morning, back dilute culture into a 50 mL YPAD at a initial OD of 0.1 OD612.

Note:
Some strains form clumps of cells - you can determine this microscopically, 20X magnification with DIC should be sufficient. A single clump of cells will only give rise to one colony on a plate, which may complicate further analysis.

3. Incubate the culture at 30^oC on a shaker at 200 rpm for around 3 to 5 hours until the OD612 reaches 0.4-0.6.

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 ddH20 and centrifuge again.

7. Pellet the cells at top speed for 15 sec and carefully remove the water. Don't touch the cell pellet.

8. Resuspend the cells to a final volume of 500 uL of 100mM LiOAc.

Note: you are looking for a fairly large size pellet in the final resuspension step. Maybe a little smaller than the size of an iPhone charger interface.

9. Boil a 1.0 ml sample of SS-DNA for 5 min. and immediately 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 52 µl samples into labelled µfuge tubes. Pellet the cells and remove water with a micropipette.

11. Make your TRAFO master mix (recipie for 1 transf.)

240 µl PEG-3350 (50% w/v)

36 µl 1.0 M. LiAc

50 µl salmon sperm ssDNA (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.

For simple deletion mutants with the precise replacement of ORFs with KanMx6 or similar constructs, setup 8x50uL PCR reactions to amplify the antibiotic marker with 50mer homology overhangs. After verifying the amplicon size, precipitate the DNA with isopropanol and resuspend in ~34uL. Use this resuspension for one yeast transformation. The efficiency of knockin will increase from ~10% to 40% if you care to generate longer homology arms (500bp - 1kb on either side). If you do generate those, combine all 3 reactions (3x8x50uL) in the isopropanol ppt step. DNA that is too clean doesn't work as well for transformations.

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 (whatever temp you were growing them normally at)

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. For FOA, plate first onto YPD, and then replica plate onto FOA. A double replica plate can help reduce background. For G418 or NAT, a single replica plating is sufficient.

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.