Sign InSign Up

Yeast Transformation - High Efficiency

Updated 7/22/2023 05:02 PM
Step-by-step
by William Shaw

Introduction

Quick and easy, high efficiency yeast transformation protocol

Materials

  • 50 % PEG-3350
    • 1M LiOAc
      • 0.1M LiOAc
        • Salmon Sperm DNA (10mg/mL)
          • ddH2O

            Procedure

            Day before

            1. Pick colony from plate into 5mL growth media for overnight growth in 30°C shaker

            Lithium Acetate Prep

            Table2
            A
            B
            C
            1
            		Number of transformations
            2
            		Volume per transformation (μL)Master mix Volume (μL)
            3
            		PEG-3350 (50% w/v) 260
            #VALUE!
            4
            		LiOAc 1.0 M 36
            #VALUE!
            5
            		ssDNA (10 mg/mL) 10
            #VALUE!
            6
            		Total 306
            #VALUE!
            7
            		Plasmid DNA + ddH2O54

            Cell prep

            1. Back dilute over night culture to OD 0.175 in 15mL growth media.
            1. Grow up yeast to OD600 ~0.75 at 30°C. Takes 4-5 hours for WT in YPD.
            1. Pellet at 2000 rpm for 10 min in large centrifuge.
            1. Prepare lithium acetate transformation while harvesting cells (see table).
            1. Wash with 0.5x volume H2O and repeat step 4.
            1. Wash with 0.5x volume 100mM LiOAc and repeat step 4.
            1. Final suspension in 0.01x volume 100mM LiOAc (100 μL per transformation - approx 300µL from 15mL culture).
            1. Pellet at 8000 rpm for 1 min at 20°C on small tabletop centrifuge.

            Heat Shock Procedure

            1. Resuspend in the lithium acetate transformation mix. I suggest resuspending with the plasmid DNA and ddH2O (54 μL) first, then mixing with the LiOAc+PEG+ssDNA mixture (306 μL) afterwards, as it is too viscous to easily disrupt the pellet.
            1. Incubate 30°C for 30 min.
            1. Incubate for a further 17 min at 42°C in water bath/heat block.
            1. Centrifuge 8000 rpm for 2 min and remove supernatant.
            1. Depending on the selection marker:

            a. Prototrophic/auxotrophy gene: Resuspend in 200 μL of H2O.

            b. Eukaryotic antibiotic: Recover in 1.0mL YPD for 2-3 hrs at 30°C.

            1. Plate desired volume on plates. Typically do half.

            Transformation of Integration Cassettes

            1. NotI digest

            - approx. 1 μg of cassette

            - 2µL NEBuffer 3.1 (NotI) or CutSmart (NotI-HF)

            - 0.2µL NotI (NEB)

            - up to 20 μL H2O

            1. 1h incubation at 37°C
            1. Optional PCR clean up for better results
            1
            Luke Andrew Lindsay
            8/3/2017 08:29 PM
            what is the clean up protocol for this?
            1. Transform the entire mixture

            Comments

            Response to: Optional PCR clean up for better results
            Luke Andrew Lindsay
            8/3/2017 08:29 PM
            what is the clean up protocol for this?
            Benchling
            Learn More
            Contact
            • Contact Us
            • 680 Folsom St, 8th Floor
              San Francisco, CA 94107
            Legal

            Benchling is actively tested against the latest versions of Chrome, Firefox, Safari, and Edge. It doesn't look like your current browser is supported - for more information, click here.