Difference between revisions of "Springer Lab: TransformationYeast"
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Revision as of 07:54, 19 July 2011
YEAST TRANSFORMATION PROTOCOL
Note: This is a simplified (i.e. sloppier) version of a protocol by Gietz. If you need to troubleshoot, refer to the more detailed original version.
This procedure takes 4-6 hours from start to finish, plus an inoculation the day before.
Besides culture media, the transformation requires the following reagents:
- Boiled salmon sperm DNA (ssDNA), 10 mg/mL
- Polyethylene glycol (PEG) 3500, 50% w/v
- Lithium acetate (LiAc), 1 M and 0.1 M
This can be boiled once and used for many transformations. Re-boil after 4-6 freeze-thaws.
- Pre-heat a heat block to 100°C.
- Boil 1 mL of ssDNA for 5 min.
- Store at -20°C and keep on ice at all times during transformation.
This can be stored at RT for a few days; otherwise make it fresh. To make 4 mL of 50% PEG:
- Measure out 2 grams of PEG 3500 into a 15 mL Falcon tube.
- Add 2 mL water and vortex. Incubate for 2 min at 42°C and vortex again.
- Note: PEG takes up a non-trivial volume when dissolved in water; this is why we start by adding only half the desired volume of water.
- Add water to 4 mL total volume. Continue to heat / vortex until all PEG dissolves.
This mix should be made fresh every transformation. A good time to make this is after day 2 step 1, when you are waiting 3-5 hours for the cells to grow. First determine how much mix you need (the table below gives recipes for 1, 5, and 10 transformations), then add the following in order, and vortex until mixed:
|Reagent||# reactions (amounts in uL)|
|1||5 (6x)||10 (11x)|
|PEG 3500 50% w/v:||240||1440||2640|
|LiAc 1.0 M||36||216||396|
|Boiled ssDNA (10 mg/ml)||10||60||110|
|Plasmid DNA + dH2O (~1ug DNA)||74||444||814|
Inoculate the yeast strain into 5 ml YPD and incubate overnight on a rotary shaker at 200 rpm and 30°C.
1. Dilute the overnight culture 1:100 into fresh media, 50 mL per 10 transformations. Incubate the flask(s) for 3-5 hours at 30°C and 200 rpm.
2. When the cells are at OD ~ 0.2-0.4 (slightly cloudy to the eye), harvest the cells by pouring into 50 mL Falcon tubes and centrifuging at 3000 g for 5 min.
3. Wash the cells once in 25 mL of sterile water, by resuspending and centrifuging again, discarding the supernatant.
4. Wash the cells once in 25 mL of 100mM LiAc.
5. Resuspend the cells in 1 mL of 100mM LiAc.
6. Transfer the cell suspension to a 1.5 mL microcentrifuge tube, centrifuge at 13000 rpm for 30 sec and discard the supernatant.
7. Add 100 mM LiAc to a final volume of 500 uL.
8. Vortex the cell suspension and aliquot 50 µl into 1.5 ml microfuge tubes, one for each transformation. Centrifuge at 13000 rpm for 30 sec and remove the supernatant.
9. Add 360 uL transformation mix to each tube and vortex for 30-60s to mix well.
11. Incubate at 42°C for 40 min.
12. Centrifuge at 13000 rpm for 30 sec and remove the transformation reagents with a pipet (or aspirator).
13. Pipette 1.0 ml of YPD into each tube; stir the pellet by with a pipet tip and vortex.
14. Incubate at 30°C for approximately 5 hours (or dilute into 5 mL YPD O/N).
15. Plate 200ul aliquots onto selective media.
16. Incubate the plates at 30°C for 3 to 4 days and count transformant colonies.
Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.