Smolke:Protocols/Yeast Media Recipes: Difference between revisions
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{{Smolke_Top}} | |||
==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 | **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 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 | ||
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**5g NH3SO4 | **5g NH3SO4 | ||
*Dissolve in 700mL Np H2O for YNB-AA NINR stock | *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) | *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%) 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 | ||
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**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) | ||
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**See here [[http://openwetware.org/wiki/Smolke:Protocols/Dropout_plates]] | **See here [[http://openwetware.org/wiki/Smolke:Protocols/Dropout_plates]] | ||
'''For yeast KanMX selection:''' | '''For yeast KanMX (G418) selection:''' | ||
** | *Cannot use ammonium sulfate as primary nitrogen source | ||
**Add 400mg/L for selection (in 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 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) | |||
-'''[[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]]) | -'''[[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 17:48, 3 October 2013
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 counter-selection:
-Michael S. Siddiqui 21:53, 4 January 2011 (EST)* 01/04/2011 (based mainly on communications from Andrew Babiskin) |