Smolke:Protocols/Yeast Media Recipes

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(New page: ==Yeast Nitrogen Base Minus Amino Acids (YNB-AA) Recipe== For 1 L Batch: 1.7g Yeast Nitrogen Base (stored in cold room) 5g NH3SO4 (Ammonium Sulfate, stored in chemical stock cabinet) D...)
(Yeast Peptone (YP) Media Recipe)
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{{Smolke_Top}}
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==Yeast Nitrogen Base Minus Amino Acids (YNB-AA) Recipe==
==Yeast Nitrogen Base Minus Amino Acids (YNB-AA) Recipe==
-
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
Autoclave (store at RT for media stocks)
Autoclave (store at RT for media stocks)
-
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: [[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
+
==Yeast Nitrogen Base Minus Amino Acids Non-Inducing, Non-Repressing (YNB-AA NINR) Recipe==
==Yeast Nitrogen Base Minus Amino Acids Non-Inducing, Non-Repressing (YNB-AA NINR) Recipe==
-
For 1 L Batch:
+
*For 1 L Batch:
-
1.7g Yeast Nitrogen Base
+
**1.7g Yeast Nitrogen Base
-
5g NH3SO4  
+
**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)
-
Dissolve in 700mL Np H2O for YNB-AA NINR stock
+
'''To use as working media:'''
-
Autoclave (store at RT for media stocks)
+
*After autoclaving add:
-
 
+
**100mL 10X (20%) Raffinose (BD 217410)
-
To use as working media:
+
**100mL 10X (10%) Sucrose (Sigma S3929)
-
After autoclaving add:
+
**100mL 10X Drop Out stock
-
100mL 10X (20%) Raffinose
+
**1 L Total Volume
-
100mL 10X (10%) Sucrose
+
-
 
+
-
100mL 10X Drop Out stock
+
-
 
+
-
1 L Total
+
==Yeast Peptone (YP) Media Recipe==
==Yeast Peptone (YP) Media Recipe==
-
For 1 L Batch:
+
*For 1 L Batch:
-
10g Yeast Extract [Bacto™ REF#212750] (stored in chemical stock cabinet)
+
**10g Yeast Extract [Bacto™ REF#212750] (stored in chemical stock cabinet)
-
20g Peptone [Bacto™ REF#211677] (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
+
**Dissolve in 900mL Np H2O for YP stock
Autoclave (store at RT for media stocks)
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 use as working media:
+
'''To pour any of these media stocks as plates:'''
-
After autoclaving add:
+
**Add 20g agar per 1L final media volume before autoclaving
-
100mL 10X (20%) Dextrose
+
**See here [[http://openwetware.org/wiki/Smolke:Protocols/Dropout_plates]]
-
1 L Total YPD (Yeast Peptone Dextrose)
+
-
To pour any of these media stocks as plates:
+
'''For yeast KanMX (G418) selection:'''
-
Add 20g agar per 1L final media volume before autoclaving
+
*Cannot use ammonium sulfate as primary nitrogen source
-
See here [[http://openwetware.org/wiki/Smolke:Protocols/Dropout_plates]]
+
*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 KanMX selection:
+
'''For yeast counter-selection:'''
-
Add 400mg/L for selection (in plates)
+
*Add 1 g/l of 5-FOA to YNB-SC solid media to counter-select for URA3
-
Add 200mg/L for maintenance (in liquid culture media)
+
**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)
-
-'''[[User:Michael S. Siddiqui|Michael S. Siddiqui]] 21:53, 4 January 2011 (EST)'''* 01/04/2011 (based mainly on communications from Andrew Babiskin)
+
-'''[[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]])

Revision as of 20:48, 3 October 2013

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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 counter-selection:

  • Add 1 g/l of 5-FOA to YNB-SC solid media to counter-select 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)

-Michael S. Siddiqui 21:53, 4 January 2011 (EST)* 01/04/2011 (based mainly on communications from Andrew Babiskin)

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