McClean: Yeast Mating Halo Assay: Difference between revisions

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==Materials==  
==Materials==  
* Item 1
* Strains of interest to check for mating type
* Item 2
* yMM421 (aka DBY7730, RC634a) MATa ade2 his6 met1 ura1 can1 cyh1 rme sst1-3
* Stock Solution 1
* yMM422 (aka DBY7442, XT1-20A) MATalpha leu- ura- ade- sst2
* Stock Solution 2
* YPD plates


==Stock Solutions==
'''Stock Solution 1'''
* This is a very simple solution, so we only need a one line description of how to make it.   
'''Stock Solution 2'''
This is a more involved solution, so we will describe how to make it in several steps:
# Step 1
# Step 2
# Step 3


==Protocol==
==Protocol==
# Step 1
* Let streaks grow 2 days 30°C.
# Step 2
* Inoculate overnight YPD cultures of the mating type testers.
# Step 3
* Dilute the overnights 1:10 with sterile media or water.
* Spread ~200 μl lawn on YPD plates, one for each mating type.
* Incubate 30°C for 30 minutes. 
* Pin your strains of interest to the lawns, or use a toothpick to make small, well-separated patches on the lawn. Make sure to flame the frogger between each plate, or use a fresh toothpick for each plate.  Do NOT pin onto tester plates thate are still wet.  This will just cause your patches to run all over the plate.
* Incubate 30°C overnight.
* Score whether or not the patch has a halo of space around it. If it does, that means that the lawn strain responded to the pheromone emitted by the patch, and thus that they are of opposite mating type. So, if a halo formed around the patched strain on the a tester plate, the strain itself is alpha. The nice thing about having both the a and the alpha tester plates is that you can double-check your scoring by making sure there is a halo on only one tester plate.




==Notes==
==Notes==
<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!  Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
*[[User:Megan N McClean|Megan N McClean]] 11:02, 3 November 2011 (EDT) We have been having some trouble lately with the yMM422 strain.  Strains of the opposite mating type have not been forming scorable halos. 
#List troubleshooting tips here.  
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
#Anecdotal observations that might be of use to others can also be posted here. 
 
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
 
==References==
==References==
Adapted from Maitreya Dunham's protocol ([http://dunham.gs.washington.edu/protocols.shtml| Dunham Lab Protocols]) which was adapted from Katja Schwartz's protocol from the Botstein lab ([http://www.princeton.edu/genomics/botstein/protocols/| Botstein Lab Protocols])
Adapted from Maitreya Dunham's protocol ([http://dunham.gs.washington.edu/protocols.shtml| Dunham Lab Protocols]) which was adapted from Katja Schwartz's protocol from the Botstein lab ([http://www.princeton.edu/genomics/botstein/protocols/| Botstein Lab Protocols])
The strains were made in the Thorner lab. See Julius et al (1983) Cell, 32, 839-52 for
documentation of DBY7730. DBY7442's exact genotype is unknown. The sst mutations
make them super-sensitive to pheromone. When exposed to pheromone of the opposite
mating type, the cells arrest.


==Contact==
==Contact==
Line 57: Line 49:
[[Category:Yeast]]
[[Category:Yeast]]
-->
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Halo Mating Type Assay
Maitreya Dunham July 2004
Protocol derived from Katja Schwartz's protocol.
In addition to your strains of interest, streak out:
DBY7730 (aka RC634a) MATa ade2 his6 met1 ura1 can1 cyh1 rme sst1-3
DBY7442 (aka XT1-20A) MATalpha leu- ura- ade- sst2
These strains were made in the Thorner lab. See Julius et al (1983) Cell, 32, 839-52 for
documentation of DBY7730. DBY7442's exact genotype is unknown. The sst mutations
make them super-sensitive to pheromone. When exposed to pheromone of the opposite
mating type, the cells arrest.
Let streaks grow 2 days 30C.
Inoculate overnight YPD cultures of the mating type testers.
Dilute the overnights 1:10 with sterile media or water.
Spread ~200 μl lawn on YPD plates, one for each mating type.
Incubate 30C 30 min.
Pin your strains of interest to the lawns, or use a toothpick to make small, well-separated
patches on the lawn. Make sure to flame the frogger between each plate, or use a fresh
toothpick for each plate.
Incubate 30C overnight.
Score whether or not the patch has a halo of space around it. If it does, that means that
the lawn strain responded to the pheromone emitted by the patch, and thus that they are
of opposite mating type. So, if a halo formed around the patched strain on the a tester
plate, the strain itself is alpha. The nice thing about having both the a and the alpha tester
plates is that you can double-check your scoring by making sure there is a halo on only
one tester plate.

Revision as of 08:02, 3 November 2011


Overview

This is a protocol for checking the mating type of a strain of interest using tester strains that are super-sensitive to mating pheromones. Your strain of interest is patched (or stamped or replica plated) onto a sensitive tester lawn and the formation of halos on the lawn (due to growth inhibition of the sensitive strain) indicates that your strain is the opposite mating type of the tester.

Materials

  • Strains of interest to check for mating type
  • yMM421 (aka DBY7730, RC634a) MATa ade2 his6 met1 ura1 can1 cyh1 rme sst1-3
  • yMM422 (aka DBY7442, XT1-20A) MATalpha leu- ura- ade- sst2
  • YPD plates


Protocol

  • Let streaks grow 2 days 30°C.
  • Inoculate overnight YPD cultures of the mating type testers.
  • Dilute the overnights 1:10 with sterile media or water.
  • Spread ~200 μl lawn on YPD plates, one for each mating type.
  • Incubate 30°C for 30 minutes.
  • Pin your strains of interest to the lawns, or use a toothpick to make small, well-separated patches on the lawn. Make sure to flame the frogger between each plate, or use a fresh toothpick for each plate. Do NOT pin onto tester plates thate are still wet. This will just cause your patches to run all over the plate.
  • Incubate 30°C overnight.
  • Score whether or not the patch has a halo of space around it. If it does, that means that the lawn strain responded to the pheromone emitted by the patch, and thus that they are of opposite mating type. So, if a halo formed around the patched strain on the a tester plate, the strain itself is alpha. The nice thing about having both the a and the alpha tester plates is that you can double-check your scoring by making sure there is a halo on only one tester plate.


Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

  • Megan N McClean 11:02, 3 November 2011 (EDT) We have been having some trouble lately with the yMM422 strain. Strains of the opposite mating type have not been forming scorable halos.

References

Adapted from Maitreya Dunham's protocol (Dunham Lab Protocols) which was adapted from Katja Schwartz's protocol from the Botstein lab (Botstein Lab Protocols)

The strains were made in the Thorner lab. See Julius et al (1983) Cell, 32, 839-52 for documentation of DBY7730. DBY7442's exact genotype is unknown. The sst mutations make them super-sensitive to pheromone. When exposed to pheromone of the opposite mating type, the cells arrest.

Contact

or instead, discuss this protocol.


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