McClean: Fixation of Yeast (P. Xu Protocol): Difference between revisions
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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 ?? | ||
*Resuspend cells in | *Resuspend cells in ?? | ||
*Store samples at 4°C until you are ready to image. | *Store samples at 4°C until you are ready to image. | ||
Revision as of 12:16, 25 July 2013
Overview
This is the protocol used by Anjali Bisaria for rapidly fixing yeast cells from chemostat cultures to image for both bud index as well as fluorescence. We found that this protocol preserves both GFP and mCherry 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 ??
- Resuspend cells in ??
- 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.