Lithium Acetate Transformation

This protocol adapted from:

Gietz, R.D. and R.A. Woods. (2002) Transformation of yeast by the Liac/SS carrier DNA/PEG method. Meth.Enzymol. 350: 87-96.

Materials

Reagent

Recipe

100mM LiAc

1.02g LiAc + water to 100ml.

Sterile filter.

1M LiAc

10.2g LiAc + water to 100ml.

Sterile filter.

50% PEG (w/v)

50g PEG3350 + 50ml H20

Autoclave at 121oC for 15 minutes

Carrier DNA

Dilute stock of sheared salmon sperm DNA to 2mg/ml in sterile water.

 Boil for 10 minutes.

Chill rapidly to make single stranded DNA. 

Store at -20oC

 

Methods

The day before you wish to perform this transformation, you must come start a 2-3ml culture of your yeast in YPD.  Incubate this culture overnight in a shaking water bath at 30oC

1.  Briefly vortex your culture, then pipette 1ml of it into a microfuge tube.

2.  Pellet the cells by spinning in a microfuge for one minute at 13,000rpm.

3.  Use a pipetman to remove the media without disturbing the cell pellet. 

4.  Resuspend the pellet 500ml of sterile water by gently pipetting the solution up and down.

5.  Pellet the cells by spinning in a microfuge for one minute at 13,000rpm.

6. Use a pipetman to remove the water without disturbing the cell pellet. 

7.  Resuspend the pellet in 200ml of 100mM lithium acetate.

8.  Pellet the cells by spinning in a microfuge for one minute at 13,000rpm.

9.  Use a pipetman to remove the lithium acetate solution without disturbing the cell pellet.

10.  Repeat steps 7, 8, and 9.

11.  Add the following ingredients on top of each cell pellet in the order listed:

            240 mL PEG3350 (50% w/v)

            36mL 1.0 M LiAc

            25mL carrier DNA (2mg/mL)

            50ml of plasmid DNA in water    

12.  Vortex the tube vigorously until the cell pellet has been completely resuspended (about 1 minute).

13.  Incubate at 30oC for 30 minutes.

14.  Heat shock sample in a water bath at 42oC for 20-25 minutes.

15.  Pellet cells by spinning in a microfuge for one minute at 13,000 rpm.

16.  Carefully remove the supernatant.

17.  Resuspend pellet in 1ml of sterile water by gently pipetting up and down.

18.  Spread 0.2ml of the cell suspension on the appropriate selective media.

19.  Pellet cells by spinning in a microfuge for one minute at 13,000 rpm.. 

20. Remove approximately 800mL of water from the tube, resuspend the pellet in the remaining liquid and plate this sample on a fresh plate containing the appropriate selective media.  Both plates should be incubated at 30oC for ~2 days.