McClean: Fixation of Yeast (McClean Protocol): Difference between revisions
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(New page: <!-- COPY EVERYTHING 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 f...) |
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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. | This is an adaptation of the protocol used by Anjali Bisaria for rapidly fixing yeast cells from chemostat cultures to image for both bud index as well as fluorescence. This protocol has been tested by Megan McClean to preserve GFP fluorescence. | ||
==Materials== | ==Materials== | ||
*Yeast cells | *Yeast cells | ||
* | *Paraformaldehyde 32% aqueous solution (Electron Microscopy Sciences Cat#15714-S) | ||
*0.1M [http://openwetware.org/wiki/McClean:_Potassium_Phosphate | Potassium Phosphate Buffer pH 7.5] | *0.1M [http://openwetware.org/wiki/McClean:_Potassium_Phosphate | Potassium Phosphate Buffer pH 7.5] | ||
==Protocol== | ==Protocol== | ||
*Prepare eppendorf tubes with 50μL of | *Prepare eppendorf tubes with 50μL of 32% paraformaldehyde, one per sample. | ||
*Add 450μL of culture to 50μL of | *Add 450μL of culture to 50μL of paraformaldehyde in an eppendorf tube and mix by inversion. (The goal is to have a final concentration of formaldehyde around 3.2%. So you can adjust the cell and paraformaldehyde volumes accordingly, as long as you end up with 3.2% paraformaldehyde). | ||
*Incubate the tube at room temperature for 15 | *Incubate the tube at room temperature for 15 minutes. | ||
*Spin down gently for 5 minutes at 8000rpm in the small eppendorf centrifuge. | *Spin down gently for 5 minutes at 8000rpm in the small eppendorf centrifuge. | ||
*Resuspend cells in | *Resuspend cells gently in 250μL of 0.1M potassium phosphate to wash any excess paraformaldehyde. | ||
*Spin down cells gently for 5 minutes at 8000rpm in the small eppendorf centrifuge. | |||
*Resuspend cells in 100μL of 0.1M potassium phosphate | |||
*Store samples at 4°C until you are ready to image. | *Store samples at 4°C until you are ready to image. | ||
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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]] 15:08, 27 May 2016 (EDT)''' This protocol is adjusted from Anjali's original protocol. Her protocol leaves out the wash step with 0.1M potassium phosphate buffer, which is very important for keeping the formaldehyde from killing GFP fluorescence. In this protocol (which was tested by MM on 5/19/16 see ntbk #8, p15) 32% paraformaldehyde was used instead of 40% formadelhyde. I (Megan) don't actually think that this makes a big difference and in the past we have found that formaldehyde actually works better. | |||
'''*[[User:Megan N McClean|Megan N McClean]] 15:08, | |||
==References== | ==References== | ||
Latest revision as of 15:02, 27 May 2016
Overview
This is an adaptation of the protocol used by Anjali Bisaria for rapidly fixing yeast cells from chemostat cultures to image for both bud index as well as fluorescence. This protocol has been tested by Megan McClean to preserve GFP fluorescence.
Materials
- Yeast cells
- Paraformaldehyde 32% aqueous solution (Electron Microscopy Sciences Cat#15714-S)
- 0.1M | Potassium Phosphate Buffer pH 7.5
Protocol
- Prepare eppendorf tubes with 50μL of 32% paraformaldehyde, one per sample.
- Add 450μL of culture to 50μL of paraformaldehyde in an eppendorf tube and mix by inversion. (The goal is to have a final concentration of formaldehyde around 3.2%. So you can adjust the cell and paraformaldehyde volumes accordingly, as long as you end up with 3.2% paraformaldehyde).
- Incubate the tube at room temperature for 15 minutes.
- Spin down gently for 5 minutes at 8000rpm in the small eppendorf centrifuge.
- Resuspend cells gently in 250μL of 0.1M potassium phosphate to wash any excess paraformaldehyde.
- Spin down cells gently for 5 minutes at 8000rpm in the small eppendorf centrifuge.
- Resuspend cells in 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, 27 May 2016 (EDT) This protocol is adjusted from Anjali's original protocol. Her protocol leaves out the wash step with 0.1M potassium phosphate buffer, which is very important for keeping the formaldehyde from killing GFP fluorescence. In this protocol (which was tested by MM on 5/19/16 see ntbk #8, p15) 32% paraformaldehyde was used instead of 40% formadelhyde. I (Megan) don't actually think that this makes a big difference and in the past we have found that formaldehyde actually works better.
References
Anjali Bisaria, Senior Thesis, 2012
Contact
Megan N McClean 3:12, 25 July 2013 (EDT)
or instead, discuss this protocol.
