McClean: Fixation of Yeast (P. Xu Protocol): Difference between revisions
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(New page: <!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL! --> ==Overview== This is the protocol used by Anjali Bisaria for rapidly fixing yeast cells from chemostat cultures to image fo...) |
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==Overview== | ==Overview== | ||
This is the protocol used by | This is the protocol used by P. Xu for fixing yeast cells for GFP fluorescence. | ||
==Materials== | ==Materials== | ||
*Yeast cells | *Yeast cells | ||
*Formaldehyde 37% (Sigma-Aldrich, #252549) | *Formaldehyde 37% (Sigma-Aldrich, #252549) | ||
*Phosphate buffered saline | |||
==Protocol== | ==Protocol== | ||
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*Add 450μL of culture to 50μL of formaldehyde in an eppendorf tube and mix by inversion. (The goal is to have a final concentration of formaldehyde around 3.7%. So you can adjust the cell and formaldehyde volumes accordingly, as long as you end up with 3.7% formaldehyde). | *Add 450μL of culture to 50μL of formaldehyde in an eppendorf tube and mix by inversion. (The goal is to have a final concentration of formaldehyde around 3.7%. So you can adjust the cell and formaldehyde volumes accordingly, as long as you end up with 3.7% formaldehyde). | ||
*Incubate the tube at room temperature for 15-20 minutes. | *Incubate the tube at room temperature for 15-20 minutes. | ||
*Spin down | *Spin down at 8000rpm for 1 minute. | ||
*Resuspend | *Wash cells with ice cold 1X PBS 3 times, spin at 8000rpm for 1 minute each time (can be longer if you need). | ||
*Resuspend cell pellet in 100ul of 1X PBS. | |||
*Store samples at 4°C until you are ready to image. | *Store samples at 4°C until you are ready to image. | ||
Latest revision as of 12:47, 25 July 2013
Overview
This is the protocol used by P. Xu for fixing yeast cells for GFP fluorescence.
Materials
- Yeast cells
- Formaldehyde 37% (Sigma-Aldrich, #252549)
- Phosphate buffered saline
Protocol
- Prepare eppendorf tubes with 50μL of 37% formaldehyde, one per sample.
- Add 450μL of culture to 50μL of formaldehyde in an eppendorf tube and mix by inversion. (The goal is to have a final concentration of formaldehyde around 3.7%. So you can adjust the cell and formaldehyde volumes accordingly, as long as you end up with 3.7% formaldehyde).
- Incubate the tube at room temperature for 15-20 minutes.
- Spin down at 8000rpm for 1 minute.
- Wash cells with ice cold 1X PBS 3 times, spin at 8000rpm for 1 minute each time (can be longer if you need).
- Resuspend cell pellet in 100ul of 1X PBS.
- Store samples at 4°C until you are ready to image.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Contact
Ping Xu 3:12, 25 July 2013 (EDT)
or instead, discuss this protocol.
