Arrest in G2/M phase using Nocodazole

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This protocol works best in YPD media. I (Samantha) tried it in SCG and it didn't work.
This protocol works best in YPD media. I (Samantha) tried it in SCG and it didn't work.
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This protocol comes from Dr. Gustavo Pesce.
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''This protocol comes from Dr. Gustavo Pesce.''
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Reagents
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'''Reagents'''
1. Make a 5x stock of YPD without glucose ("5x YP"). Store a bunch of 100 ml bottles of this at room temperature.  
1. Make a 5x stock of YPD without glucose ("5x YP"). Store a bunch of 100 ml bottles of this at room temperature.  
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3. Make a 10 mg/ml stock of Nocodazole in DMSO, store at -20.
3. Make a 10 mg/ml stock of Nocodazole in DMSO, store at -20.
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Protocol
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'''Protocol'''
1. Grow up cells overnight in log phase.
1. Grow up cells overnight in log phase.

Revision as of 12:49, 31 May 2005

This protocol works best in YPD media. I (Samantha) tried it in SCG and it didn't work.

This protocol comes from Dr. Gustavo Pesce.

Reagents

1. Make a 5x stock of YPD without glucose ("5x YP"). Store a bunch of 100 ml bottles of this at room temperature.

2. Make a 30 mg/ml stock of Benomyl in DMSO, store at -20 degrees C.

3. Make a 10 mg/ml stock of Nocodazole in DMSO, store at -20.


Protocol

1. Grow up cells overnight in log phase.

2. The day of your experiment, prepare 1x YP with this stock and sterile water. Bring it to a boil in microwave, and then immediately add Benomyl to 10 µg/ml. Let it sit until it cools to 20-40 ºC.

2. Add glucose. You can store the media for a week or so at 4 ºC or use it immediately.

3. In parallel, prepare another batch without benomyl. This will be your control. Boil it and all, but add only DMSO to it.

4. Add nocodazole to the benomyl media to 15 µg/ml. Add an equal amount of DMSO to the control.

5. Add cells from step 1 into the nocodazole/benomyl and control media to a OD600 of 0.5. After 2 hours almost all cells appear like the number "8", arrested in metaphase. They will stay like that for ever, growing in size and not dividing. Some will show new buds.

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