McClean: Fixation of Yeast (P. Xu Protocol)

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(Overview)
(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 cells in ??
+
*Wash cells with 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.

Revision as of 14:44, 25 July 2013


Contents

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 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.

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