Difference between revisions of "Springer Lab: TransformationYeast"
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This can be stored at RT for a few days
This can be stored at RT for a few days. mL of PEG
* Measure out
* Measure out grams of PEG 3500 into a mL .
** Note: PEG takes up
* Add mL water and vortex . for min.
* Add water to
** Note: PEG takes up volume when dissolved in water; this is why we start by adding only half the desired volume of water.
* Add water to mL total volume. Continue to /
Latest revision as of 07:49, 22 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, or several weeks if in an airtight container such as a glass bottle (NOT Falcon tube). The following recipe is for 50 mL of PEG, which is good for ~200 transformations. Smaller amounts can be made by adjusting the amounts accordingly.
- Measure out 25 grams of PEG 3500 into a 100 mL glass bottle.
- Add 25 mL water and vortex well. Let rest for 10 min.
- Note: PEG takes up volume when dissolved in water; this is why we start by adding only half the desired volume of water.
- Add water to 50 mL total volume. Vortex again and let rest until solution looks clear. Continue to vortex / let rest if necessary.
- Sterilize by autoclave on liquid 20 minute cycle or sterile filter into a new bottle.
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|
1. Inoculate the yeast strain into 5 ml YPD and incubate overnight on a rotary shaker at 200 rpm and 30°C. Also incubate a flask of 50 mL YPD for every 10 transformations you will do.
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 ~ 0.3-0.5 (moderately 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. This last mixing is very important.
10. Incubate at 30°C for 30 min.
11. Heat shock at 42°C for 15 min. (This time might vary depending on strain. Refer to the heat shock duration page for information about specific strains.)
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 5 hours or O/N.
15. Plate 200ul aliquots onto selective media. If cells have settled, mix by pipetting up and down before plating.
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.