McClean: Cycloheximide Stock Solution

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(New page: <!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL! --> ==Overview== This is the standard 50mg/ml stock solution. This is diluted to 100μg/mL to inhibit translation (though you ...)
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==Procedure==
==Procedure==
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Shake the new vial of α-factor to get all of the power on the bottom of the vialRemove the lid and pipet 1ml of DMSO into the vial of α-factor from SigmaPipet 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.
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Weigh out the appropriate amount of cycloheximide and add it to the appropriate amount of DMSO to get a 50mg/ml solutionUse the Botstein lab's balance as it is much more accurate than oursIt seems to make the most sense to aim for about 100mg (but if it is slightly over or under that is fine) and then put this in a 2ml eppendorf or similar tube (you can use a 15ml conical if you are going to make a larger volume) and add the appropriate amount of DMSO to this based on what you weighed out.
==Notes==
==Notes==

Revision as of 11:01, 6 October 2011


Contents

Overview

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.


Materials

  • Cycloheximide (MP Biomedicals, Cat #100183)
  • DMSO (Fluka 41639, Ultra for molecular biology; stored in the Flammables cabinet)


Procedure

Weigh out the appropriate amount of cycloheximide and add it to the appropriate amount of DMSO to get a 50mg/ml solution. Use the Botstein lab's balance as it is much more accurate than ours. It seems to make the most sense to aim for about 100mg (but if it is slightly over or under that is fine) and then put this in a 2ml eppendorf or similar tube (you can use a 15ml conical if you are going to make a larger volume) and add the appropriate amount of DMSO to this based on what you weighed out.

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 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.

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