High Efficiency Transformation-PDF

Protocol

Day 0

• Make sure you have the necessary solutions (instructions for how to make them can be found here):
  1. Single-stranded carrier DNA
  2. PEG 3350 50% w/vol
  3. 1.0 M LiAc
• Make sure you have enough plasmid DNA for your transformations. Each transformation should use between 0.5 and 1.0 µg of plasmid.

Day 1

Inoculate the yeast strain into 5 ml of liquid medium and incubate overnight.

Day 2

1. Determine the titer of the yeast culture by pipetting 10 µl of cells into 1.0 ml of water in a spectrophotometer cuvette and measuring the OD at 600 nm. For many yeast strains a suspension containing 1 x 10⁶ cells/ml will give an OD₆₀₀ of 0.1.
2. Transfer 50 ml of YPD to the pre-warmed culture flask and add 2.5 x 10⁸ cells to give 5 x 10⁶ cells/ml.
3. Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.
4. Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells (the next step).
   • 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.
5. When the cell titer is at least 2 x 10⁷ cells/ml, which should take about 4 hours, harvest the cells by centrifugation at 3000 g for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of sterile water.
   • If the cell titer is higher/lower than 2×10⁷ cells/ml, you’ll dilute it back down/up in a subsequent step
   • Now would also be a good time to start warming up the 42°C water bath you’ll need later on
6. Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge at 3000 g for 30 sec and discard the supernatant.
7. Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.
   • If your cell titer was higher/lower than 2×10⁷ cells/ml, add proportionally more/less water at this step. Also, note that if you split your initial 50ml of culture into multiple tubes, the water should be split appropriately between the tubes.
8. Pipette 100 µl samples (ca. 10⁸ cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at 3000g for 30 sec and remove the supernatant.
9. Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.

It’s generally easiest to make a master mix of PEG, LiAc and the SS-carrier DNA and add 326 µl from the master mix to each transformation tube before adding the plasmid DNA + water component.

For the plasmid DNA + water component, you’ll need to calculate, for each transformation, what vol. of your plasmid DNA solution contain the appropriate amount of plasmid DNA (0.5-1.0 µg). Add in the appropriate amount of plasmid DNA solution and then make it up to 34µl with water.

	Number of Transformations
Reagents	1	5 (6X)	10 (11X)
PEG 3350 50% w/v	240 µl	1440 µl	2640 µl
LiAc 1.0 M	36 µl	216 µl	396 µl
Boiled SS-carrier DNA	50 µl	300 µl	550 µl
Plasmid DNA plus Water	34 µl	204 µl	374 µl
Total	360 µl	2160 µl	3960 µl

1. Add 360 µl of Transformation Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.
2. Incubate the tubes in a 42°C water bath for 40 min.
3. Microcentrifuge at 3000 g for 30 sec and remove the Transformation Mix with a micropipettor.
4. Pipette 1.0 ml of sterile water into each tube; stir the pellet by with a micropipette tip and vortex .
   • We like to be a gentle as possible at this step if high efficiency is important. Excessive washing washes away transformants.
5. Plate appropriate dilutions of the cell suspension onto SC selection medium. For transformation with an integrating plasmid (YIp), linear construct or oligonucleotide, plate 200 µl onto each of 5 plates; for a YEp, YRp or YCp library plasmid dilute 10 µl of the suspension into 1.0 ml of water and plate 10 and 100 µl samples of the dilute onto two plates each. The 10 µl samples should be pipetted directly into 100 µl puddles of sterile water on the SC selection medium.