McClean: Sporulation: Difference between revisions
(→Notes) |
No edit summary |
||
(4 intermediate revisions by the same user not shown) | |||
Line 3: | Line 3: | ||
==Overview== | ==Overview== | ||
To sporulate yeast cells need to be starved for nitrogen in the presence of a nonfermentable carbon source. This protocol uses acetate as the carbon source. | |||
==Materials== | ==Materials== | ||
* 1% Potassium Acetate (autoclaved) | * 1% Potassium Acetate (autoclaved) | ||
Line 31: | Line 30: | ||
# Incubate at room temperature on a roller wheel for 1 day and then transfer to 30°C for 2 more days (can incubate longer, if desired). | # Incubate at room temperature on a roller wheel for 1 day and then transfer to 30°C for 2 more days (can incubate longer, if desired). | ||
# Check culture for spore formation on day 2 and continue checking until you see tetrads. | # Check culture for spore formation on day 2 and continue checking until you see tetrads. | ||
==Notes== | ==Notes== | ||
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! | Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! | ||
#'''*[[User:Megan N McClean|Megan N McClean]] 13:25, 30 September 2011 (EDT)''': According to the Botstein lab, when sporulating, if diploid is homozygous for an auxotrophic mutation, add that nutrient to the potassium acetate (about ¼ the amount listed for addition to SD media… any more and it could be used as a nitrogen source). You may need to play with the concentration of nutrient that you add. So far, the McClean lab has only sporulated prototrophic strains using this protocol, so the first person to try it/get it to work with an auxotrophic strain, please leave a note here! | #'''*[[User:Megan N McClean|Megan N McClean]] 13:25, 30 September 2011 (EDT)''': According to the Botstein lab, when sporulating, if diploid is homozygous for an auxotrophic mutation, add that nutrient to the potassium acetate (about ¼ the amount listed for addition to SD media… any more and it could be used as a nitrogen source). You may need to play with the concentration of nutrient that you add. So far, the McClean lab has only sporulated prototrophic strains using this protocol, so the first person to try it/get it to work with an auxotrophic strain, please leave a note here! | ||
#'''*[[User:Megan N McClean|Megan N McClean]] 13:25, 30 September 2011 (EDT)''': To sporulate yeast cells need to be starved for nitrogen in the presence of a nonfermentable | #'''*[[User:Megan N McClean|Megan N McClean]] 13:25, 30 September 2011 (EDT)''': To sporulate yeast cells need to be starved for nitrogen in the presence of a nonfermentable carbon source. | ||
carbon source. | |||
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | ||
==References== | ==References== | ||
Adapted from the Botstein lab protocol available at: http://www.princeton.edu/genomics/botstein/protocols/Sporulation_and_Tetrad_Dissection.pdf | |||
==Contact== | ==Contact== | ||
'''*[[User:Megan N McClean|Megan N McClean]] 13:29, 30 September 2011 (EDT)''' | |||
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | ||
<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | <!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | ||
[[Category:Yeast]] | |||
[[Category:Protocol]] | |||
<!-- Move the relevant categories above this line to tag your protocol with the label | <!-- Move the relevant categories above this line to tag your protocol with the label | ||
[[Category:Media]] | [[Category:Media]] | ||
--> | --> |
Revision as of 07:39, 31 October 2011
Overview
To sporulate yeast cells need to be starved for nitrogen in the presence of a nonfermentable carbon source. This protocol uses acetate as the carbon source.
Materials
- 1% Potassium Acetate (autoclaved)
SPORULATION
Mark Hickman uses this protocol and regularly has sporulation efficiencies of >50%.
1. Grow a 5 mL overnight culture; dilute 1:50 in the morning and grow for 4 hours at 30°C (to log
phase).
2. Pellet cells, wash in 1 mL of 1% potassium acetate, and resuspend in 3-4 mL of 1%
potassium acetate.
3. Incubate at room temperature on a roller wheel for 3 day (can incubate longer, if desired).
NOTE: When sporulating, if diploid is homozygous for an auxotrophic mutation, add that nutrient
to the potassium acetate (about ¼ the amount listed for addition to SD media… any more and it
could be used as a nitrogen source).
Stock Solutions
1% Potassium Acetate
Protocol
- Grow a 5 mL overnight culture; dilute 1:50 in the morning and grow for 4 hours at 30°C (to log
phase).
- Pellet cells, wash in 1 mL of 1% potassium acetate, and resuspend in 3-4 mL of 1%
potassium acetate.
- Incubate at room temperature on a roller wheel for 1 day and then transfer to 30°C for 2 more days (can incubate longer, if desired).
- Check culture for spore formation on day 2 and continue checking until you see tetrads.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- *Megan N McClean 13:25, 30 September 2011 (EDT): According to the Botstein lab, when sporulating, if diploid is homozygous for an auxotrophic mutation, add that nutrient to the potassium acetate (about ¼ the amount listed for addition to SD media… any more and it could be used as a nitrogen source). You may need to play with the concentration of nutrient that you add. So far, the McClean lab has only sporulated prototrophic strains using this protocol, so the first person to try it/get it to work with an auxotrophic strain, please leave a note here!
- *Megan N McClean 13:25, 30 September 2011 (EDT): To sporulate yeast cells need to be starved for nitrogen in the presence of a nonfermentable carbon source.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Adapted from the Botstein lab protocol available at: http://www.princeton.edu/genomics/botstein/protocols/Sporulation_and_Tetrad_Dissection.pdf
Contact
*Megan N McClean 13:29, 30 September 2011 (EDT) or instead, discuss this protocol.