Springer Lab: TransformationYeast

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(Transformation mix)
(previous version had incorrect heat shock duration.)
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==Day 1==
==Day 1==
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Inoculate the yeast strain into 5 ml YPD and incubate overnight on a rotary shaker at 200 rpm and 30°C.
+
'''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.
==Day 2==
==Day 2==
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'''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.
'''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.
+
'''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.
'''3.''' Wash the cells once in 25 mL of sterile water, by resuspending and centrifuging again, discarding the supernatant.
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'''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.
'''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|transformation mix]] to each tube and vortex for 30-60s to mix well.
+
'''9.''' Add 360 uL [[#Transformation mix|transformation mix]] to each tube and '''vortex for 30-60s to mix well'''. This last mixing is very important.
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'''11.''' Incubate at 42°C for 40 min.
+
'''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).
'''12.''' Centrifuge at 13000 rpm for 30 sec and remove the transformation reagents with a pipet (or aspirator).
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'''13.''' Pipette 1.0 ml of YPD into each tube; stir the pellet by with a pipet tip and vortex.
'''13.''' Pipette 1.0 ml of YPD into each tube; stir the pellet by with a pipet tip and vortex.
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'''14.''' Incubate at 30°C for approximately 5 hours (or dilute into 5 mL YPD O/N).
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'''14.''' Incubate at 30°C for 5 hours or O/N.
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'''15.''' Plate 200ul aliquots onto selective media.  
+
'''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.
'''16.''' Incubate the plates at 30°C for 3 to 4 days and count transformant colonies.

Revision as of 09:44, 22 July 2011

Back to Springer Lab | Last updated by Jue Wang 10:53, 19 July 2011 (EDT)

Contents

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.

Reagents

Besides culture media, the transformation requires the following reagents:

  • Icebox
  • Boiled salmon sperm DNA (ssDNA), 10 mg/mL
  • Polyethylene glycol (PEG) 3500, 50% w/v
  • Lithium acetate (LiAc), 1 M and 0.1 M

Boiled ssDNA

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.

50% PEG

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.

Transformation mix

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
Total 360 2160 3960

Day 1

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.

Day 2

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.

References

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.

http://home.cc.umanitoba.ca/~gietz/method.html

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