Pichia Adenine Dropout Agar, PAD Agar | 250g

Pichia Adenine Dropout (PAD) Agar, 250 g

Production

This media is prepared in small batches and will be prepared after the purchase, taking around 5 to 10 business days for handling.

Application

For selecting ADE2 gene-deficient Pichia Pastoris colonies.

Why use PAD rather than minimal media for ADE-deficient Pichia selection?

Although minimal media (MY), minimal media with glycerol/methanol (MGY/MM) and complex media (CY) can also be used to select ADE mutant colonies. However, MY and even CY are much slower and less efficient at picking out white/red colonies compared to PAD media because Pichia growth on MY is slow (small colonies are difficult to compare visually) and CY contains yeast extract, and yeast extract contains adenine, so all colonies initially appear the same white, making it difficult to achieve high-resolution colour assays (the contrast between pure white/white/pink/red) between colonies. Besides, MY & CY are a bit complex to prepare because of the complex trace elements stock & vitamins stock need to be prepared separately.

Components, per litre

Preparation

[Standard preparation, recommended] 

1.       Prepare 0.5 litre of 10X SD stock solution: Dissolve 167 g mixture A in distilled or Milli-Q water and mix well, make it to 500 ml.  Sterilise by 0.22µm filtration (recommended) or autoclave at 115℃ for 30 minutes.  Store the stock at room temperature. The shelf life of this solution is approximately one year.

2.       Weigh 1.22 g of mixture B, and a suitable amount of agar (1~2% w/v recommended), in distilled or Milli-Q Water, make it to 900ml. Sterilise by autoclaving at 121°C for 15 minutes.

3.       Add 100 mL of sterile 10X SD stock into the freshly autoclaved (no longer boiling) mixture B solution, mix well and pour the plates while hot.  Store the PAD agar plates at 4°C. The shelf life is several months.

[Fast preparation, may cause a dark colour of the agar]

1.       Weigh 33.4 g of mixture A (SD medium), 1.22 g of mixture B, and a suitable amount of Agar ^[1] in distilled or Milli-Q Water, make it to 1000ml.

2.       Sterilise by autoclaving at 121℃ for 15 minutes ^[2]. Aliquot as needed.

[1]     1~2% w/v recommended

[2]     If equipment allows, autoclave at 115℃ for 30 minutes to prevent the darkening of the solution due to a Maillard reaction on glucose catalysed by ammonium sulphate and heat.