Yeast Transformation Protocol
Introduction
Materials
- YPD Media
- 2% glucose
- Adenine
- Carbenicillin
- Polyethylene glycol (PEG)
- Lithium Acetate (LiOAc)
- Carrier DNA (Single-Stranded)
- Plasmid of Interest
- Linearized
Procedure
- Grow an overnight culture of yeast.
Pipet 4mL of appropriate media into a culture tube (or use 3mL in a block) NOTE: Begin as close to 5 pm as possible but err on the later side.
With pipette tip, transfer yeast cells from a colony to the flask - Don't shy away from picking a lot of cells.
Attach pipette to the tip and fluff solution
Leave in 30 degree shaker incubator overnight
The next morning, test the OD:
Pipette 300µL of media and yeast into a flat bottom plate and place in plate reader - you can omit media as control
(new plate reader file and 96 well costar clear (no *))
- Outgrow in appropriate media. (Starting OD ~0.1, Volume ~3mL/transformation)
300µL-400µL of yeast culture of ~0.1 OD/transformation and 3mL of YPAD/transformation. Err on the side of 400µL to begin transformation at 3 or 4PM
- Incubate in 30oC shaker until OD ~0.6-0.8
Begin YPAD outgrow at 9-9:30AM
Check OD at 2PM and 4PM
This usually means leaving it in the shaker for 4-6 hours
Check OD of the yeast by pipetting 300mL into a flat bottom plate and placing in plate reader
- Just before pulling culture, prepare master mix:
Material Material | Volume per reaction Volume per reaction | |
1 1 | 50% PEG | 240 uL |
2 2 | 1M LiOAc | 36 uL |
3 3 | SS-DNA (Carrier DNA) | 25 uL |
4 4 | Linearized Plasmid | ~ 1.5 ug |
Preparing SS-DNA (salmon sperm DNA):
boil in hot bath for 5 min
keep on ice for 5 min
Preparing Plasmid: refer to Linearizing Plasmids for Yeast Transformation
- Pellet (centrifuge) culture in 15mL conical tubes (5 min, 1000g (setting #3)). Resuspend (fluff) in 100µL sterile H2O/transformation.
- Add 100µL cells to ~300µL master mix (in an epi tube) Vortex softly.
- Heat shock cells in 42oC water bath for 42 min. (Very time sensitive)
- Pellet cells (5 min, 1000g) (small centrifuge)
- Wash step (optional): Add 300µL of appropriate media (SD -URA if the product strains have URA homology, YPD if product strains have Hygro resistance)
Resuspend pellet in media
Pellet cells again, 5 min @ 1000g
Pour off media, then proceed to step 10
- Resuspend in 125µL of appropriate media.** Only for auxotrophic markers like URA and LEU
Otherwise:
In a sterile block,
If rescuing for 2 hours:
add 250µL of YPAD
add cells that have been resuspended in 50µL of YPAD
If rescuing overnight:
add 3mL of YPD
add cells that have been resuspended in 50µL of YPD (Split cells across 3-4 wells of the block. Pellet and resuspend in the morning.)
Put block in 30 degree shaker (should put paper towels around a corner to provide a better fit)
- Plate cells and incubate at 30oC.
Flatten tip on lid of the plate and spread media thinly and wait to dry
Leave in incubator for 2 days
This is Meghan Bragdon's protocol for transforming yeast! Originally written by Khalil lab, 06/29/2012. The numbered steps are from this original, all descriptions are Wetlab notes.