This recipe will make 10 aliquots of cells for transformation. Note that it takes a substantial amount of time for yeast to grow in Step 3, so it is often convenient to start the protocol early in the morning and complete the transformation in the afternoon/evening.
1. Grow ~5-6 mL overnight yeast cells in 2xYPAD for >12-16h at 30C 300rpm until cultures have reached saturation.
2. Add 5 mL of overnight cultures into 50 mL of 2xYPAD in a 250 mL bottle. Record the OD600 of the solution (remember to calculate the OD600(corrected) = OD600 of cells - OD600 of 2xYPAD blank). This will likely be around OD600(corrected) ~ 0.1. Often 2xYPAD has an OD600 of about 0.2.
3. Grow at 30C 300 rpm until OD600(corrected) ~ 0.4-0.5. This will equate to about 2 cell divisions, which is necessary for good transformation efficiency. This can take 6-10 hours.
4. Harvest cells by centrifugation at 3000g for 5 minutes (in a 50 mL tube) and resuspend the cell pellet in 25 mL of sterile ddH2O.
5. Repeat - harvest cells by centrifugation at 3000g for 5 minutes and resuspend the cell pellet in 25 mL of sterile ddH2O.
6. Repeat - harvest cells by centrifugation at 3000g for 5 minutes and resuspend the cell pellet in 1.0 mL of sterile ddH2O.
7. Transfer cells to a 1.5 mL microcentrifuge tube and centrifuge at 13000g for 30 seconds. Discard the supernatant and resuspend cells in 1.0 mL of sterile ddH2O.
8. Pipette 100 uL aliquots into microcentrifuge tubes, one for each transformation. Centrifuge for 13000g for 30 seconds and remove the supernatant.
9. Denature aliquots of single-stranded carrier DNA (salmon sperm DNA) by placing them in a boiling water bath for 5 minutes and immediately chill in an ice/water bath. Denatured carrier DNA can be boiled three or four times without significant loss of activity.
10. Prepare the transformation mix and resuspend the cells by pipetting and intermittent vortexing. This can be done directly in the microcentrifuge tubes
Transformation Mix Component |
Volume |
|---|---|
PEG 3350 (50% w/v) |
240 uL |
Lithium Acetate (1.0 M) |
36 uL |
Single-stranded carrier DNA (2.0 mg/mL), denatured in a boiling water bath |
50 uL |
DNA to transform (linearized plasmid for integration into the chromosome or autonomously replicating plasmid) |
Up to 34 uL total |
ddH2O |
34 uL - (uL added of DNA to transform) |
Total |
360 uL |
11. Place the tubes in a 42C water bath for 45 minutes.
12. Centrifuge the tubes at 13000g for 30 seconds and remove the supernatant.
A. For prototrophic gene selection: Pipette 200 uL - 1.0 ml of sterile water into the transformation tube. Stir the pellet with a sterile micropipette tip to break the cell pellet and then vortex mix to uniformly resuspend pellet. I usually resuspend in 200 uL.
B. For eukaryotic antibiotic gene selection: Pipette 200 uL - 1.0 ml of YPD liquid medium into the transformation tube. Vortex mix to resuspend pellet. Incubate for 2--3 h at 30C to ensure good expression from the input plasmid DNA.
13. Plate 200 uL of the cell suspension on the appropriate plates. Incubate plates at 30C for 2-3 days.
1 Comment
Unknown User (sele@mit.edu)
The OD600 reading value indicated is that of a 10x diluted culture as you need for measurement and not the actual OD600 of the culture itself.