McClean: Alpha Factor Stock

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Current revision (11:58, 6 October 2011) (view source)
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Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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'''*[[User:Megan N McClean|Megan N McClean]]''': Make sure to keep track of lot numbers (write it down in your notebook, on your stock solutions, and each time you do an experiment with α-factor).
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'''*[[User:Megan N McClean|Megan N McClean]]''': Make sure to keep track of lot numbers (write it down in your notebook, on your stock solutions, and each time you do an experiment with α-factor). '''Really'''.  Please do this!
'''*[[User:Megan N McClean|Megan N McClean]]''': I generally find it useful to make a series of 1:10 dilutions of the α-factor (1mg/mL, 100μg/mL, 10μg/mL, 1μg/mL) when I first make the stock, as these are 1000x the concentrations I generally start out with for most experiments (so that I can add 1μL of stock solution per ml of media).  I usually make 900μL of all of these dilute stocks (ie, start with 100μL of 1mg/ml stock +900μL of DMSO for the 100μg/mL stock, from that take 100μL and add it to another 900μL of DMSO for the 10μg/mL, and then once more for the 1μg/mL stock).  I then divide the 900μL into 100μL aliquots so that I don't have to freeze/thaw the entire stock too often.   
'''*[[User:Megan N McClean|Megan N McClean]]''': I generally find it useful to make a series of 1:10 dilutions of the α-factor (1mg/mL, 100μg/mL, 10μg/mL, 1μg/mL) when I first make the stock, as these are 1000x the concentrations I generally start out with for most experiments (so that I can add 1μL of stock solution per ml of media).  I usually make 900μL of all of these dilute stocks (ie, start with 100μL of 1mg/ml stock +900μL of DMSO for the 100μg/mL stock, from that take 100μL and add it to another 900μL of DMSO for the 10μg/mL, and then once more for the 1μg/mL stock).  I then divide the 900μL into 100μL aliquots so that I don't have to freeze/thaw the entire stock too often.   

Current revision


Contents

Overview

This is the standard 1mg/ml stock solution we use for inducing the pheromone pathway in budding yeast. Generally, we find that the different batches of pheromone can be somewhat variable so it is a good idea to make up a stock that will last you through your experiment and keep it in your own -20°C box. If you have to switch stocks mid-experiment you need to do some calibration experiments to make sure the activity is the same.

Materials

  • α1-Mating Factor acetate salt (Sigma T6901-1mg)
  • DMSO (Fluka 41639, Ultra for molecular biology; stored in the Flammables cabinet)


Procedure

Shake the new vial of α-factor to get all of the power on the bottom of the vial. Remove the lid and pipet 1ml of DMSO into the vial of α-factor from Sigma. Pipet up and down and put the lid back on an swirl to dissolve all of the alpha-factor into the DMSO. Then remove the liquid and aliquot it in 100μL eppendorfs into individual eppendorfs and store these in your own -20°C box. Keep track of the lot # of the pheromone both with your stock solutions and when you do your experiments.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

*Megan N McClean: Make sure to keep track of lot numbers (write it down in your notebook, on your stock solutions, and each time you do an experiment with α-factor). Really. Please do this!

*Megan N McClean: I generally find it useful to make a series of 1:10 dilutions of the α-factor (1mg/mL, 100μg/mL, 10μg/mL, 1μg/mL) when I first make the stock, as these are 1000x the concentrations I generally start out with for most experiments (so that I can add 1μL of stock solution per ml of media). I usually make 900μL of all of these dilute stocks (ie, start with 100μL of 1mg/ml stock +900μL of DMSO for the 100μg/mL stock, from that take 100μL and add it to another 900μL of DMSO for the 10μg/mL, and then once more for the 1μg/mL stock). I then divide the 900μL into 100μL aliquots so that I don't have to freeze/thaw the entire stock too often.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

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