Smolke:Protocols/Yeast Media Recipes: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(6 intermediate revisions by 2 users not shown) | |||
Line 4: | Line 4: | ||
*For 1 L Batch: | *For 1 L Batch: | ||
**1.7g Yeast Nitrogen Base (stored in cold room) | **1.7g Yeast Nitrogen Base (stored in cold room) | ||
**5g (NH4)2SO4 (Ammonium Sulfate, stored in chemical stock cabinet) | **5g (NH4)2SO4 (Ammonium Sulfate, stored in chemical stock cabinet, EMD | ||
168355) | |||
*Dissolve in 800mL Np H2O for YNB-AA stock | *Dissolve in 800mL Np H2O for YNB-AA stock | ||
*Adjust pH value to 5.8 using NaOH (starting May 2011) -Yen-Hsiang | *Adjust pH value to 5.8 using NaOH (starting May 2011) -Yen-Hsiang | ||
Line 11: | Line 12: | ||
'''To use as working media:''' | '''To use as working media:''' | ||
*After autoclaving add: | *After autoclaving add: | ||
**100mL 10X (20%) Dextrose | **100mL 10X (20%) Dextrose (BD 215530) | ||
**100mL 10X Drop Out stock (10X DO stock recipe here: [[http://openwetware.org/wiki/Smolke:Protocols/DO_Media]]) | **100mL 10X Drop Out stock (10X DO stock recipe here: [[http://openwetware.org/wiki/Smolke:Protocols/DO_Media]]) | ||
**1 L Total Volume | **1 L Total Volume | ||
Line 25: | Line 26: | ||
'''To use as working media:''' | '''To use as working media:''' | ||
*After autoclaving add: | *After autoclaving add: | ||
**100mL 10X (20%) Raffinose | **100mL 10X (20%) Raffinose (BD 217410) | ||
**100mL 10X (10%) Sucrose | **100mL 10X (10%) Sucrose (Sigma S3929) | ||
**100mL 10X Drop Out stock | **100mL 10X Drop Out stock | ||
**1 L Total Volume | **1 L Total Volume | ||
Line 48: | Line 49: | ||
'''For yeast KanMX (G418) selection:''' | '''For yeast KanMX (G418) selection:''' | ||
*Cannot use ammonium sulfate as primary nitrogen source | *Cannot use ammonium sulfate as primary nitrogen source | ||
*Add G418 to YP or to YNB with 1 g/ | *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 400mg/L for selection (in plates) | ||
*Add 200mg/L for maintenance (in liquid culture media) | *Add 200mg/L for maintenance (in liquid culture media) | ||
-'''[[User:Michael S. Siddiqui|Michael S. Siddiqui]] 21:53, 4 January 2011 (EST)'''* 01/04/2011 ( | '''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 | |||
**dissolve 2.6 grams into 200 ml, filter sterilize, add 50ml per 500ml sleeve of plates | |||
**if stored cold or frozen, will crash out of solution; heating helps dissolve | |||
**detailed useful compound info here: http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/f7129pis.pdf | |||
*For alpha-AAD/LYS2,5 counterselection replace nitrogen source with 2g/L (0.2%) D,L-alpha-aminoadipate (Bachem, F-1185) | |||
**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 | |||
-'''[[User:Michael S. Siddiqui|Michael S. Siddiqui]] 21:53, 4 January 2011 (EST)'''* 01/04/2011 (aided by [[User:Andrewb|Andrew Babiskin]]) |
Latest revision as of 14:46, 18 March 2014
Yeast Nitrogen Base Minus Amino Acids (YNB-AA) Recipe
168355)
Autoclave (store at RT for media stocks) To use as working media:
Yeast Nitrogen Base Minus Amino Acids Non-Inducing, Non-Repressing (YNB-AA NINR) Recipe
To use as working media:
Yeast Peptone (YP) Media Recipe
Autoclave (store at RT for media stocks) To use as working media: After autoclaving add:
To pour any of these media stocks as plates:
For yeast KanMX (G418) selection:
For yeast counterselection:
-Michael S. Siddiqui 21:53, 4 January 2011 (EST)* 01/04/2011 (aided by Andrew Babiskin) |