Springer Lab: TransformationYeast

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=YEAST TRANSFORMATION PROTOCOL=
 
Back to [[Springer Lab]] | Last updated by [[User:Jue Wang|Jue Wang]] 10:53, 19 July 2011 (EDT)
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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'''Note:''' This is a simplified (i.e. sloppier) version of a protocol by Gietz. If you need to troubleshoot, refer to the more detailed version [http://home.cc.umanitoba.ca/~gietz/method.html 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:
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
* Icebox
* Boiled salmon sperm DNA (ssDNA), 2 mL/mL ([[#Appendix: Preparing boiled ssDNA|instructions]])
* 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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* Polyethylene glycol (PEG) 3500, 50% w/v ([[Appendix: Preparing 50% PEG|instructions]])
* Lithium acetate (LiAc), 1 M and 0.1 M
* Lithium acetate (LiAc), 1 M and 0.1 M
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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.
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'''2.''' Harvest the cells by pouring into 50 mL Falcon tubes and centrifuging at 3000 g for 5 min.
'''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.
'''3.''' Wash the cells once in 25 mL of sterile water, by resuspending and centrifuging again, discarding the supernatant.
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'''4.''' Wash the cells once in 25 mL of 100mM LiAc.
'''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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'''5.''' Resuspend the cells in 1 mL of 100mM LiAc.
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'''5.''' Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells.
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'''6.''' Transfer the cell suspension to a 1.5 mL microcentrifuge tube, centrifuge at 13000 rpm for 30 sec and discard the supernatant.
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* 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.****
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'''7.''' Add 100 mM LiAc to a final volume of 500 uL.
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'''6.''' Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard 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.
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'''7.''' Add 100 mM LiAc to a final volume of 500 ul.
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'''9.''' Add the following reagents to each transformation '''''in order''''':
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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.
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{|
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|PEG 3500 50% w/v: |   240 µl
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|-
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|LiAc 1.0 M | 36 µl
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|-
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|Boiled SS-carrier DNA (2 mg/ml) | 50 µl
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|-
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|Plasmid DNA plus Water (use 0.1 - 10 ug tranforming DNA) | 34 µl
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|}
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'''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.
 
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'''9.''' Add the following reagents to each transformation in the order given:
 
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**PEG 3500 50% w/v:   240 µl
 
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**LiAc 1.0 M: 36 µl
 
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**Boiled SS-carrier DNA (2 mg/ml):   50 µl
 
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**Plasmid DNA plus Water (use 0.1 - 10 mg tranforming DNA): 34 µl
 
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==Appendix: Preparing boiled ssDNA==
==Appendix: Preparing boiled ssDNA==
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==Appendix: Preparing 50% PEG 3500==
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'''5.''' Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells.
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* 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.****
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 +
 
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==Appendix: Preparing 50% PEG==
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 11:04, 19 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 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.

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 the following reagents to each transformation in order:

  240 µl
36 µl
50 µl
 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

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.****


Appendix: Preparing 50% PEG

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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