Yeast Nitrogen Base Minus Amino Acids (YNB-AA) Recipe
- For 1 L Batch:
- 1.7g Yeast Nitrogen Base (stored in cold room)
- 5g (NH4)2SO4 (Ammonium Sulfate, stored in chemical stock cabinet, EMD
168355)
- Dissolve in 800mL Np H2O for YNB-AA stock
- Adjust pH value to 5.8 using NaOH (starting May 2011) -Yen-Hsiang
Autoclave (store at RT for media stocks)
To use as working media:
- After autoclaving add:
- 100mL 10X (20%) Dextrose (BD 215530)
- 100mL 10X Drop Out stock (10X DO stock recipe here: [[1]])
- 1 L Total Volume
Yeast Nitrogen Base Minus Amino Acids Non-Inducing, Non-Repressing (YNB-AA NINR) Recipe
- For 1 L Batch:
- 1.7g Yeast Nitrogen Base
- 5g NH3SO4
- Dissolve in 700mL Np H2O for YNB-AA NINR stock
- Adjust pH value to 5.8 using NaOH (starting May 2011) -Yen-Hsiang
- Autoclave (store at RT for media stocks)
To use as working media:
- After autoclaving add:
- 100mL 10X (20%) Raffinose (BD 217410)
- 100mL 10X (10%) Sucrose (Sigma S3929)
- 100mL 10X Drop Out stock
- 1 L Total Volume
Yeast Peptone (YP) Media Recipe
- For 1 L Batch:
- 10g Yeast Extract [Bacto™ REF#212750] (stored in chemical stock cabinet)
- 20g Peptone [Bacto™ REF#211677] (stored in chemical stock cabinet)
- 80 mg of Adenine hemisulfate (Sigma Chemical Co. Ltd., cat. no. A-3159) (stored in chemical stock cabinet)
- Dissolve in 900mL Np H2O for YP stock
Autoclave (store at RT for media stocks)
To use as working media:
After autoclaving add:
- 100mL 10X (20%) Dextrose
- 1 L Total Volume YPD (Yeast Peptone Dextrose)
To pour any of these media stocks as plates:
- Add 20g agar per 1L final media volume before autoclaving
- See here [[2]]
For yeast KanMX (G418) selection:
- Cannot use ammonium sulfate as primary nitrogen source
- Add G418 to YP or to YNB with 1 g/L MSG (Sigma G1626) instead of ammounium sulfate
- Aqueous solutions stored frozen at -20 to -70 °C should be stable for approximately six months.
- Powder G418 sulfate (Geneticin) is stored in chemical room.
- Add 400mg/L for selection (in plates)
- Add 200mg/L for maintenance (in liquid culture media)
For yeast counterselection:
- Add 1 g/l of 5-FOA to YNB-SC solid media to counterselect for URA3
- While adding 20% dextrose and 10x SC AA mix, have 5-FOA dissolved in DMSO and add before stirring and pouring out agar (for a typical single sleeve 500ml media batch, this can be 500mg of 5-FOA in 5ml DMSO)
- Add 1.3 g/l of 5-fluorocytosine (5-FC) to YNB-SC solid media to counterselect for FCY1
- For alpha-AAD/LYS2,5 counterselection replace nitrogen source with 2g/L (0.2%) D,L-alpha-aminoadipate
- dissolve 1 gram alpha-AAD in 20ml of water and pH with KOH to pH 6.0 (otherwise insoluble), filter sterilize and add to YNB with special AA mix
- two recipes have been shown to work for this counterselection
- YNB-alpha-AAD + URA,TRP,HIS,LEU,LYS
- YNB-alpha-AAD + URA,TRP,HIS,LEU,LYS,MET,ADE
- special media informed by Gottschling lab site: http://labs.fhcrc.org/gottschling/Yeast%20Protocols/lore.html
-Michael S. Siddiqui 21:53, 4 January 2011 (EST)* 01/04/2011 (aided by Andrew Babiskin)
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