Editing Pichia EXpression (section)

=== '''Reagents and Solutions'''  ===

<br>The following reagents and solutions are required for all four LiAc/SS-DNA/PEG protocols.<br><u>Lithium Acetate (1.0 M)</u>. Dissolve 5.1 g of lithium acetate dihydrate (Sigma Chemical Co. Ltd., St. Louis, MO) in 50 ml of water, sterilize by autoclaving for 15 min, and store at room temperature. 

<br><u>Polyethylene Glycol 3350 (50%, w/v)</u>. Dissolve 50 g of PEG 3350 (Sigma) in 30 ml of distilled, deionized water in a 150-ml beaker on a stirring hot plate. When the solution has cooled to room temperature, make the volume up to 100-ml in a 100-ml measuring cylinder and mix thoroughly by inversion. Pour the solution in to a suitable glass bottle and autoclave for 15 min. Store, securely capped, at room<br>temperature. Evaporation of water from the solution will increase the concentration of PEG and severely reduce the yield of transformants.<br>Single-Stranded Carrier DNA (2.0 mg/ml). Dissolve 200 mg of salmon sperm DNA (Sigma) in 100 ml of TE buffer (10 mM Tris-HCl, 1 mM Na2 EDTA, pH 8.0) by stirring at 4 ° for 1-2 hr. Store 1.0-ml samples at - 2 0 °. Denature the carrier DNA in a boiling water bath for 5 min and chill in ice/water before use. Boiled samples, stored at - 2 0 °, can be reused or reboiled three times without loss of activity. 

<u>Yeast Growth Media</u>. Yeast strains are grown up on plates of YPAD agar (YpD13 supplemented with 100 mg adenine hemisulfate per liter). The yeast cells to be transformed are usually regrown in liquid 2× YPAD medium 3 (2% Bacto-yeast extract, 4% Bacto-peptone, 4% glucose and adenine hemisulfate, 100 mg/liter). 

<br><u>SC selection medium 12</u> is adjusted to pH 5.6 with 1.0 N NaOH and autoclaved. Commercial formulations of YPD agar (Bacto YPD agar) and broth (Bacto YPD broth) media are available from Becton Dickinson Microbiology Systems (Becton Dickinson, Sparks, MD; www.bd.com). These media should be supplemented with adenine hemisulfate as above.