McClean: Fixation of Yeast (P. Xu Protocol)

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Protocol)
Current revision (14:47, 25 July 2013) (view source)
(Materials)
 
(3 intermediate revisions not shown.)
Line 2: Line 2:
==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.
+
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??
+
*Phosphate buffered saline
==Protocol==
==Protocol==
Line 13: Line 13:
*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 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.

Current revision


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 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.

Personal tools