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Large scale yeast chemical transformation

Goal

Perform high efficient yeast transformation with LiAc method.

Procedure

  1. Grow a single colony, which was grown for 2 days on YPD plate at 30 ℃, in 3 mL YPD+1xcarb at 30 ℃, 8.5 speed for overnight.

  2. Dilute the ON culture by 50 fold in 50 mL YPD (2% glucose + 1xcarb) and grow them in a 250 ml flask at 30 ℃, 200rpm for \~4 hours.

  3. Prepare 5x 1 µg of the linearized plasmid by cutting with a specific enzyme for a couple of hours.

  4. After 4 hours (\~2 doublings) pellet cells at 3,000g, 1 min, room temp.

  5. Wash cells twice with 50 ml room temp milliQ H2O, resuspending each time followed by pelleting as above.

  6. Denature 250μl (per transformation) of 2mg/ml Salmon Sperm DNA in TE at 99°C for 5 min, and chill immediately in an ice-water bath.

  7. Make 5x 326μl of transformation mix per 50 ml transformation (include negative control):

    Reagent and Stock Concentration Volume 5 reactions
    50% wt/vol PEG 3350 240μl 1.2 ml
    1M Lithium Acetate 36μl 180 μl
    2mg/ml Carrier DNA 50μl 250 μl
    Total 326μl 1.63 ml

  8. Resuspend cells by pipetting several times in the above transformation mix.

    1. Add 1.63mL of transformation buffer into cell pellets then resuspend well.

  9. Prepare 5x 24 µl (5x 1 µg in ddH2O) of linearized plasmids in 5x 1.5 ml tubes.

  10. Add 326 µl of transformation buffer into each 1.5 ml tube with linealized plasmids (or no DAN as -ve control) and mix well.

  11. Incubate at 42°C, 40 min. Flick the tube couple of times every 15 min to prevent cells from settling down.

  12. Plate 100μl and 10 µl on SCD-URA plates. Incubate at 30°C for 2-3 days.

  13. If we need single colonies, we can keep the 10 µl or 100 µl tranformation plates and stop here.

  14. If we need a highly efficient transformation for "barcoded yeast pools", grow remained samples in 50 mL of SCD-selection media in 250mL of Flask at 30 ℃, 200rpm for overnight (\~20 hours).

  15. Dilute the overnight cultures (OD600\~2) in 100 fold then regrow them in the same condition (50 ml SCD-selection in 250 ml flask, 200rpm at 30 ℃) for one more overnight (\~20 hours).

  16. We expect 300-400 colonies from 10 µl plating. So liquid culture in SCD-selection should include initialy around 50,000-60,000 colonies

  17. We can make 5 × 1 ml glycerol stocks from the culture in SCD-selection liquid culture after the second dilution.

Reference