Springer Lab: TransformationYeast

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Back to [[Springer Lab]]
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Back to [[Springer Lab]] | Last updated by [[User:Jue Wang|Jue Wang]] 10:53, 19 July 2011 (EDT)
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=YEAST TRANSFORMATION PROTOCOL=
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'''Note:''' This is a simplified (i.e. sloppier) version of the protocol by Gietz. For troubleshooting and/or higher efficiency, refer to the more detailed version [http://home.cc.umanitoba.ca/~gietz/method.html here].
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'''YEAST TRANSFORMATION PROTOCOL'''
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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, for which recipes and instructions can be found at the bottom of the page:
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* Icebox
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* Boiled salmon sperm DNA (ssDNA), 2 mL/mL ([[#Appendix: Preparing boiled ssDNA|instructions]])
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* Polyethylene glycol (PEG) 3500, 50% w/v (instructions)
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* Lithium acetate (LiAc), 1 M and 0.1 M
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Last updated 20110629
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==Day 1==
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'''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.
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Inoculate the yeast strain into 5 ml of liquid medium (2x YPAD or SC selection medium) and incubate overnight on a rotary shaker at 200 rpm and 30°C. Place a bottle of double strength YPAD broth (2x YPAD) and a 250 ml culture flask in the incubator as well.
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==Day 2==
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'''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.
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'''1.''' Determine the titer of the yeast culture by pipetting 10 ul of cells into 1.0 ml of water in a spectrophotometer cuvette and measuring the OD at 600 nm. For many yeast strains a suspension containing 1 x 10^6 cells/ml will give an OD600 of 0.1.
 
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'''2.''' Transfer 50 ml of the pre-warmed 2x YPAD to the pre-warmed culture flask and add 2.5 x 10^8 cells to give 5 x 10^6 cells/ml.
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'''2.''' Harvest the cells by pouring into 50 mL Falcon tubes and centrifuging at 3000 g for 5 min.
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'''3.''' Wash the cells once in 25 mL of sterile water, by resuspending and centrifuging again, discarding the supernatant.
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'''3.''' Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.
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'''4.''' Wash the cells once in 25 mL of 100mM LiAc.
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'''5.''' Resuspend the cells in 1 mL of 100mM LiAc and transfer to a 1.5 mL microcentrifuge tube.
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Note:
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i) It is important to allow the cells to complete at least two divisions.
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ii)This will take 3 to 5 hours.
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iii)This culture will give sufficient cells for 10 transformations.
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iv) Transformation efficiency (transformants/ µg plasmid/108 cells) remains constant for 3 to 4 celldivisions.
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Note: In practice, we dilute 0/N culture 1:100 and let it grow for 3-5 hours.
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Checklist: ssDNA, PEG, LiAc (1M and 0.1M), icebox
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'''4.''' When the cell titer is at least 2 x 10^7 cells/ml, which should take about 4 hours, harvest the cells by centrifugation at 3000 g for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of 100mM LiAc.
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'''15.''' Incubate the plates at 30°C for 3 to 4 days and count the number of transformants.
'''15.''' Incubate the plates at 30°C for 3 to 4 days and count the number of transformants.
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==Appendix: Preparing boiled ssDNA==
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==Appendix: Preparing 50% PEG 3500==
Modified from: 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.
Modified from: 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
http://home.cc.umanitoba.ca/~gietz/method.html

Revision as of 09:53, 19 July 2011

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

YEAST TRANSFORMATION PROTOCOL

Note: This is a simplified (i.e. sloppier) version of the protocol by Gietz. For troubleshooting and/or higher efficiency, refer to the more detailed version here.

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, for which recipes and instructions can be found at the bottom of the page:

  • Icebox
  • Boiled salmon sperm DNA (ssDNA), 2 mL/mL (instructions)
  • Polyethylene glycol (PEG) 3500, 50% w/v (instructions)
  • Lithium acetate (LiAc), 1 M and 0.1 M

Day 1

Inoculate the yeast strain into 5 ml YPD and incubate overnight on a rotary shaker at 200 rpm and 30°C.

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. 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 and transfer to a 1.5 mL microcentrifuge tube.


5. Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells.

  • It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your own freezer box and boil after 3-4 freeze-thaws. But keep on ice when out.****


6. Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.


7. Add 100 mM LiAc to a final volume of 500 ul. Note:If the cell titer of the culture is greater than 2 x 10^7 cells/ml the volume then increase the volume to maintain the titer of this suspension at 2 x 10^9 cells/ml. If the titer of the culture is less than 2 x 10^7 cells/ml then decrease volume.


8. Vortex the cell suspension and pipette 50 µl samples (ca. 10^8 cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at top speed for 30 sec and remove the supernatant.


9. Add the following reagents to each transformation in the order given:

    • PEG 3500 50% w/v:   240 µl
    • LiAc 1.0 M: 36 µl
    • Boiled SS-carrier DNA (2 mg/ml):   50 µl
    • Plasmid DNA plus Water (use 0.1 - 10 mg tranforming DNA): 34 µl


10. Incubate for 30 minutes at 30°C

Checklist: 42'C heating block or water bath

11. Incubate the tubes in a 42°C water bath for 20-25 min. Note:The optimum time can vary for different yeast strains. Please test this if you need high efficiency from your transformations.


12. Microcentrifuge at top speed for 30 sec and remove the transformation reagents with a micropipettor.


13. Pipette 1.0 ml of YPD into each tube; stir the pellet by with a micropipette tip and vortex . 1. Note:We like to be a gentle as possible at this step if high efficiency is important. Excessive washing washes away transformants!!!!


14. Incubate at 30°C for approximately 5 hours, then plate 200ul aliquots onto selective plates.


15. Incubate the plates at 30°C for 3 to 4 days and count the number of transformants.

Appendix: Preparing boiled ssDNA

Appendix: Preparing 50% PEG 3500

Modified from: 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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