YEAST MEDIA PREPARATIONS

 

YPD

Bacto-yeast extract - 10 gm

Bacto-peptone - 20 gm

Bacto-agar - 25 gm

Add enough water to bring the volume upto 900 mL.

Autoclave for 20 min.

Add 100 mL 20% filtered dextrose and mix.

Pour plates.

Note:- For YPD media, do not add the bacto-agar.

2) SD medium:-

Bacto-yeast nitrogen base - 1.7 gm

(w/o amino acids & ammonium sulfate)

Ammonium sulfate - 5 gm

Bacto-agar - 25 gm

Add enough water to bring the volume upto 800 mL.

Autoclave for 20 min.

Add the following:-

Filtered 20% Dextrose - 100 mL

10x Amino acid mix - 100 mL.

NOTE:- For gal/raf medium, add 100 mL 0f 10x gal/raf i.e. 20% galactose (Sigma G-250) + 10% raffinose (Sigma R-750).

 

 

 

10x AMINO ACID MIX (L-Amino acids):-

To 800 mL of dH2O, add the following:-

Ade - 0.3 g Lys - 0.5 g Leu - 0.5 g Pro - 0.3 g

Ura - 0.3 g His - 0.3 g Phe - 0.6 g Ser - 4.0 g

Tyr - 0.6 g Met - 0.3 g Asp - 1.2 g Val - 1.5 g

Trp - 0.3 g Arg - 0.3 g Thr - 2.0 g Ile - 0.3 g

Glu - 1.0 g

 

Yeast Transformation

For a standard yeast transformation, miniprep DNA purified on columns or extracted with phenol/chloroform can be used. For 10 -15 independent transformations, a 100 mL culture is enough.

Inoculate yeast strain into 5 mL YPD. Grow at 30o C with vigorous shaking (250 rpm) O/N.

Add to 100 mL prewarmed YPD medium such that the OD600 is about 0.1 - 0.2. Grow at 30 oC with vigorous shaking till the OD600 reaches 0.5

Transfer to 2 50 mL tubes and spin at 3K for 5 min. at room temperature.

Discard the supernatant and resuspend the pellet in 5 mL TE/Li. Pool the pellets and spin in a 15 mL tube at 3K for 5 min.

Discard the supernatant and resuspend the pellet in 5 mL TE/Li. Incubate at 30oC for 30 min. with slow shaking.

Spin for 3 min. at 3K, discard the supernatant and resuspend the pellet in 1 mL. TE/Li.

Aliquot 100 ul. cells into each eppendorf tube and add 1-2 ug. of plasmid DNA to it. Also add ss carrier DNA to every sample.