McClean: Cycloheximide Stock Solution
This is the standard 50mg/ml stock solution. This is diluted to 100μg/mL to inhibit translation (though you should check this concentration does what you think it does whenever you develop a new protocol using cycloheximide). Each person in the lab makes up their own stock of cycloheximide and keeps it in their own -20°C box.
- Cycloheximide (MP Biomedicals, Cat #100183)
- DMSO (Fluka 41639, Ultra for molecular biology; stored in the Flammables cabinet)
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.
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 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.
- Megan N McClean 14:01, 6 October 2011 (EDT)
or instead, discuss this protocol.