McClean: Fixation of Yeast (Bisaria Protocol): Difference between revisions

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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...)
 
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<!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL!  -->
<!-- COPY EVERYTHING BELOW HERE TO START YOUR OWN PROTOCOL!  -->


==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 preserved both GFP and mCherry fluorescence well.
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==
*HotMaster Taq Polymerase
*Yeast cells
*10x HotMaster Buffer with Mg<sup>2+</sup>
*Formaldehyde 37% (Sigma-Aldrich, #252549)
**The polymerase and buffer come in the 5 Prime kit FP220320 ordered from Fisher
*0.1M Potassium Phosphate
*10mM dNTPs
*Forward primer (10μM)
*Reverse primer (10μM)
*Sterile H<sub>2</sub>O


==Protocol==
==Protocol==
 
*Prepare eppendorf tubes with 50μL of 37% formaldehyde, one per sample.
Add approximately 0.6μL of cells (tiny amount) with the tip of a sterile toothpick into the bottom of PCR tubes or plate.  Once you've put cells into the PCR vessel, put the end of the toothpick into ~100μL sterile YPD in either an eppendorf or the well of a culture plate.  You will use this to inoculate an overnight culture if your colony PCR works.  Keep the eppendorfs or culture plate at either room temperature or 30°C while you run the PCR, either is fine. 
*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.
Microwave cells in the PCR tube/plate for 1min (2X).  Put microwaved cells on ice.   
*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.
Add the reaction mix (described below) to the PCR tube/plate.  It is recommended to make up a master mix if you are doing multiple colonies.  Put the PCR tubes/plate into the thermocycler and run the Colony PCR program described below.
 
===PCR Reaction Mix===
 
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| 10x HotMaster Taq Buffer with Mg2+ ||5μL
|-
| 10mM dNTP mix ||1μL
|-
| 10μM Primer 1|| 1μL
|-
| 10μM Primer 2 ||1μL
|-
| HotMaster Taq DNA polymerase ||0.5μL
|-
| Sterile Water|| 40.5μL
|-
| style="background:#f0f0f0;"| Total Volume
|  style="background:#f0f0f0;"| 50μL
|}
 
===PCR Program===
Run PCR on Colony PCR program :
*95°C 4min
*For the following steps, reduce the temperature ‐1°C each cycle and cycle 30x's
**94°C 1min
**65°C 1min
**68°C 2min
*For the following steps, cycle 30x's
**94°C 30sec
**50°C 30sec
**72°C 1min
*72°C 5min
*4°C Forever
**Please don't leave the thermocycler running at 4°C for longer than an hour or so, it wears out the machine.  If you need to leave your PCR for longer, please change the last step of the program so that instead of holding at 4°C the program just ends (letting the samples come to room temperature).  Letting the sample come to room temperature, even overnight, does not seem to cause any problems for the DNA.
 
 
 
 
==='Freezing Down Positive Transformants'===
Once you have run the colony PCR and confirmed which colonies are correct and which are not, take the 100μL of YPD that you set aside before, inoculate it into 4mls of fresh YPD in a test tube, and grow it to saturation overnight.  Use this to freeze down glycerol stocks of the strain the following morning.


==Notes==
==Notes==
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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]]''' The lengthy touchdown PCR program protocol was devised by Megan in the early days of the McClean lab when we were struggling with getting colony PCR to work reproducibly.  The program is probably WAY longer and more complicated than it needs to be (and ties up the thermocycler for 4 hours at a time) so it would be worth someone's time to figure out a shorter program. Once you do that, please add it as another colony PCR protocol on openwetware and add a note here.
'''*[[User:Stephanie S. Steltzer|Stephanie S. Steltzer]] 11:03, 3 August 2015''' After troubleshooting we found to preserve the GFP fluorescence best, incubate the tube for just 15min at room temp and after it is spun down, wash once in KPO4 (~0.5-1ml) before resuspending cells in 75-100μL of 0.1M KPO4.Also, cells must be imaged day of.


'''*[[User:Megan N McClean|Megan N McClean]]''' We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions.  If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.
'''*[[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]]''' 15:36, 25 March 2013 (EDT) See [http://openwetware.org/wiki/McClean:_Sequencing_Colony_PCR_Product Sequencing Colony PCR Product] for how I prep my colony PCR products for sequencing
'''*[[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==
Botstein Lab protocols: http://www.princeton.edu/genomics/botstein/protocols/colony_PCR.htm
Anjali Bisaria, Senior Thesis, 2012


==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 09:20, 3 August 2015


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.

*Stephanie S. Steltzer 11:03, 3 August 2015 After troubleshooting we found to preserve the GFP fluorescence best, incubate the tube for just 15min at room temp and after it is spun down, wash once in KPO4 (~0.5-1ml) before resuspending cells in 75-100μL of 0.1M KPO4.Also, cells must be imaged day of.

*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.


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