Smolke:Protocols/Yeast Media Recipes

From OpenWetWare

< Smolke:Protocols(Difference between revisions)
Jump to: navigation, search
(Yeast Peptone (YP) Media Recipe)
Current revision (17:46, 18 March 2014) (view source)
(Yeast Peptone (YP) Media Recipe)
 
(8 intermediate revisions 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 NH3SO4 (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/l MSG instead of ammounium sulfate
+
*Add G418 to YP or to YNB with 1 g/L MSG (Sigma G1626) instead of ammounium sulfate
-
*Frozen stock is good for 3 months.
+
*Aqueous solutions stored frozen at -20 to -70 °C should be stable for approximately six months.  
-
*dry G418 is stored in chemical room.
+
*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 (based mainly on communications from [[User:Andrewb|Andrew Babiskin]]) and Dan Kvitek
+
'''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]])

Current revision

Home        Contact        Internal        Protocols        Lab Members        Publications        Research       


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 (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

-Michael S. Siddiqui 21:53, 4 January 2011 (EST)* 01/04/2011 (aided by Andrew Babiskin)

Personal tools