McClean: Fixation of Yeast (Bisaria Protocol): Difference between revisions
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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 protocol | 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== | ==Materials== | ||
*Yeast cells | *Yeast cells | ||
*Formaldehyde 37% (Sigma-Aldrich, #252549) | *Formaldehyde 37% (Sigma-Aldrich, #252549) | ||
*0.1M Potassium Phosphate | |||
==Protocol== | ==Protocol== | ||
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Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | ||
'''*[[User:Megan N McClean|Megan N McClean]]''' | '''*[[User:Megan N McClean|Megan N McClean]] 15:08, 25 July 2013 (EDT)''' We found empirically that removing the formaldehyde is very important for preserving GFP fluorescence. Don't incubate cells with formaldehyde for more than 20 minutes. | ||
'''*[[User:Megan N McClean|Megan N McClean]]''' | '''*[[User:Megan N McClean|Megan N McClean]]''' I have also used the Koshland Lab protocol ([http://mcb.berkeley.edu/labs/koshland/Protocols/MICROSCOPY/gfpfix.html GFP Fixation]) and found that it works approximately as well as this protocol. | ||
==References== | ==References== | ||
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==Contact== | ==Contact== | ||
'''[[User:Megan N McClean|Megan N McClean]] ''' | '''[[User:Megan N McClean|Megan N McClean]] 3:12, 25 July 2013 (EDT)''' | ||
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | ||
Revision as of 12:22, 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)
- 0.1M Potassium Phosphate
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 gently for 5 minutes at 8000rpm in the small eppendorf centrifuge.
- Resuspend cells in 75-100μL of 0.1M potassium phosphate
- 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.
*Megan N McClean 15:08, 25 July 2013 (EDT) We found empirically that removing the formaldehyde is very important for preserving GFP fluorescence. Don't incubate cells with formaldehyde for more than 20 minutes.
*Megan N McClean I have also used the Koshland Lab protocol (GFP Fixation) and found that it works approximately as well as this protocol.
References
Anjali Bisaria, Senior Thesis, 2012
Contact
Megan N McClean 3:12, 25 July 2013 (EDT)
or instead, discuss this protocol.
