Editing Pichia EXpression (section)

== Recipes<br><br>  ==

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'''20% Dextrose'''<br>(10X)<br>To prepare 1 liter of 20% Dextrose (10X) stock solution:<br>1. Dissolve the contents of the Dextrose pouch from the PichiaPink Media Kit in 1000 mL of distilled water.<br>2. Autoclave for 15 minutes or filter sterilize.<br>Store at room temperature. The shelf life of this solution is approximately one year.<br> 

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'''10X YNB''' (13.4% Yeast Nitrogen Base with Ammonium Sulfate, without amino acids)<br>Dissolve 134 g of yeast nitrogen base (YNB) with ammonium sulfate and without amino acids in 1000 mL of water and filter sterilize. Heat the solution to dissolve YNB completely in water. Store at 4°C. Alternatively, use 34 g of YNB without ammonium sulfate and amino acids and 100 g of ammonium sulfate. The shelf life of this solution is approximately one year. If you are using the YNB pouch included in the kit, follow the directions on the pouch.<br>Note: Pichia cells exhibit optimal growth with higher YNB concentrations, therefore, the amount of YNB used in this kit is twice as concentrated as YNB formulations for Saccharomyces.<br><br> 

'''50X B (0.02% Biotin)'''<br>Dissolve 20 mg biotin in 100 mL of water and filter sterilize. Store at 4°C. The shelf life of this solution is approximately one year.<br><br> 

'''100X A (0.2% Adenine)'''<br>Dissolve 200 mg of L-adenine in 100 mL of water. Heat the solution, if necessary, to no greater than 50°C in order to dissolve. Filter sterilize and store at 4°C. The shelf life of this solution is approximately one year.<br> 

<br>'''10X D (20% Dextrose)'''<br>Dissolve 200 g of D-glucose in 1000 mL of water. Autoclave for 15 minutes, or filter sterilize. The shelf life of this solution is approximately one year.<br><br> 

'''10X M (5% Methanol)'''<br>Mix 5 mL of methanol with 95 mL of water. Filter sterilize and store at 4°C. The shelf life of this solution is approximately two months.<br><br> 

'''10X GY (10% Glycerol) '''<br>Mix 100 mL of glycerol with 900 mL of water. Filter sterilize or autoclave. Store at room temperature. The shelf life of this solution is greater than one year.<br><br> 

'''1 M potassium phosphate buffer, pH 6.0:'''<br>Combine 132 mL of 1 M K<sub>2</sub>HPO<sub>4</sub>, 868 mL of 1 M KH<sub>2</sub>PO<sub>4</sub> and confirm that the pH is 6.0 ± 0.1 (if the pH needs to be adjusted, use phosphoric acid or KOH). Sterilize by autoclaving and store at room temperature. The shelf life of this solution is greater than one year.<br><br> 

'''Buffered Glycerol-complex Medium (BMGY) and Buffered Methanol-complex Medium''' (BMMY)<br>(1 liter)<br>1% yeast extract<br>2% peptone<br>100 mM potassium phosphate, pH 6.0<br>1.34% YNB<br>0.0004% biotin<br>1% glycerol or 0.5% methanol<br>1. Dissolve 10 g of yeast extract, 20 g peptone in 700 mL water.<br>2. Autoclave 20 minutes on liquid cycle.<br>3. Cool to room temperature, then add the following and mix well:<br>100 mL 1 M potassium phosphate buffer, pH 6.0<br>100 mL 10X YNB<br>20 mL 50X B<br>100 mL 10X GY<br>4.<br>For BMMY, add 100 mL 10X M instead of glycerol.<br>Store media at 4°C. The shelf life of this solution is approximately two months<br><br> 

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'''Minimal Glycerol Medium ± Adenine (1 liter)'''<br>1.34% YNB<br>1% glycerol<br>0.0004% biotin<br>± 0.002% Adenine<br>1. Combine aseptically 800 mL autoclaved water with 100 mL of 10X YNB, 20 mL of 50X B, and 100 mL of 10X GY.<br>2. For growth of ade2 strains in this medium, a version can be made that contains adenine (called MGYA) by adding 10 mL of 100X A stock solution.<br>Store at 4°C. The shelf life of this solution is approximately two months.<br><br> 

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[[Category:Protocols]]