Difference between revisions of "McClean: Fixation of Yeast (P. Xu Protocol)"

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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...)
 
(Materials)
 
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==Overview==
 
==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.
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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)
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*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 gently for 5 minutes at 8000rpm in the small eppendorf centrifuge.
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*Spin down at 8000rpm for 1 minute.
*Resuspend cells in 75-100μL of 0.1M potassium phosphate
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*Wash cells with ice cold 1X PBS 3 times, spin at 8000rpm for 1 minute each time (can be longer if you need).
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*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 11:47, 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.