McClean: Alpha Factor Stock: Difference between revisions
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==Overview== | ==Overview== | ||
This is the standard 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. | 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== | ==Materials== | ||
α1-Mating Factor acetate salt (Sigma T6901-1mg) | *α1-Mating Factor acetate salt (Sigma T6901-1mg) | ||
DMSO (Fluka 41639, Ultra for molecular biology; stored in the Flammables cabinet) | *DMSO (Fluka 41639, Ultra for molecular biology; stored in the Flammables cabinet) | ||
==Procedure== | ==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. | |||
==Notes== | ==Notes== | ||
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! | ||
'''*[[User:Megan N McClean|Megan N McClean]]''': I generally find it useful to make a series 1:10 dilutions of the α-factor (1mg/m1, 100μg/mL, 10μg/mL, 1μg/mL) when I first make the stock, as these are the concentrations I generally start out with for most experiments. I usually make 900μL of all of these dilute stocks (ie, start with 100μL of 1mg/ml stock +900μL of DMSO, from that take 100 | |||
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. | ||
Revision as of 08:48, 6 October 2011
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.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! *Megan N McClean: I generally find it useful to make a series 1:10 dilutions of the α-factor (1mg/m1, 100μg/mL, 10μg/mL, 1μg/mL) when I first make the stock, as these are the concentrations I generally start out with for most experiments. I usually make 900μL of all of these dilute stocks (ie, start with 100μL of 1mg/ml stock +900μL of DMSO, from that take 100 Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
Contact
- Megan N McClean 14:01, 6 October 2011 (EDT)
or instead, discuss this protocol.
