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12.5: Exercise 1 - Yeast transformation

  • Page ID
    17570
  • The following protocol is a slight modification of the “Quick and Dirty” transformation protocol described by Amberg et al. (2005). With careful attention to detail and cooperative strains, this procedure can yield thousands of transformants per μg plasmid DNA. Modifications to this method can increase its efficiency by several orders of magnitude (Gietz and Schiestl, 2007), which would be required if linear pieces of DNA were being used to tranform yeast.

    Prepare a transformation master mix

    1. Prepare a transformation master mix. The following ingredients provide enough reagents for five transformation reactions. Combine and mix in a microcentrifuge tube:

    100 μL sterile 2 M lithium acetate (freshly prepared)
    400 μL sterile 50% PEG-3350
    4 μL 2-mercaptoethanol (STINKY!! add this in the fume hood!)

    Set up individual transformation reactions - for each transformation:

    2. Add 15 μL of the denatured salmon sperm DNA (2 mg/mL) to a new microcentrifuge tubelabeled with the name (or code) of the plasmid.

    Note: It is important for the salmon sperm DNA to be single-stranded for this procedure to work well. Boil the DNA for 5 minutes to denature the DNA. Quick chill the DNA by placing it immediately on ice. Keep the DNA on ice until you are ready to use it.

    3. Add 5 μL of miniprep plasmid DNA to the appropriately labeled microcentrifuge tube.

    4. Add 100 μL of transformation mix from step 1 to each microcentrifuge tube. Vortex for 10-15 seconds to mix the contents.

    5. Using a sterile toothpick or micropipette tip, scrape a large yeast colony (or the equivalent of a “match head” of yeast) from a YPD plate. Transfer the yeast to the microcentrifuge tube containing the transformation/DNA solution (step 4) by twirling the toothpick several times. Be sure that the cells are uniformly suspended before proceeding.

    Repeat steps 2-5 for each of your transformation reactions. Be sure to include a control that contains no plasmid DNA.

    6. Incubate the transformation mixtures at 37 ̊C with shaking for 30-45 minutes.

    Plate the transformed cells on selective media lacking uracil.

    7. Remove 10 μL of the resuspended cells and add them to 90 μL of sterile water in a microcentrifuge tube. This sample will be serially diluted for a spot plate (step 9) that you will use to calculate the transformation efficiency.

    8. Spread the remainder of the mixture on a selective media plate lacking uracil. Transfer the transformation reaction to the plate, and then shake out ~4 sterile glass beads that will spread the cells. Cover the plates and spend 0.5-1 minutes agitating the plates so that the beads spread the transformation mixture evenly over the surface of the plate. Discard the glass beads into the appropriate waste containers, so they can be sterilized and used again. Incubate the plates at 30 ̊C until colonies can be detected. The earliest that colonies will be visible is usually 2 days. If the colonies are small, allow them to grow an additional day(s) at 30 ̊C. Count the number of colonies on the plate.

    Determine the number of viable cells in the transformation mixture.

    9. Prepare a series of 4 additional dilutions of the cells set aside in step 7. Use these dilutions
    for a spot plate on YPD media. Each row on the plate should contain cells from a different transformation reaction. Incubate the cells at 30 ̊C or room temperature until individual colonies can be detected. Do not allow the plate to overgrow, because you need to distinguish individual colonies.

    Calculate the transformation efficiency. The efficiency of transformation is influenced by both the quality of the DNA used and the precise details of the transformation procedure.

    10. Calculate the fraction of cells that were transformed as shown below. The total volume of transformation mixture was ~120 μL, including yeast cells. Ten μL was used for spot plating and the remaining 100 μL was used for the transformation.

    12-efficiency.jpg

    11. Transformation efficiencies are usually expressed by the number of cells transformed per μg DNA. In the last lab (Chapter 11), you analyzed your plasmid preparations on agarose gels and obtained a rough estimate of the DNA concentrations of your plasmid preparations. Note that you analyzed 7 μL of plasmid prep on those gels. In this transformation lab, you used 5 μL of your plasmid preps.

    Calculate the transformation efficiency:

    A. Multiply that number of transformed cells on the YC-plate by 1.1
    (Only 100 out of 110 μL in the transformation reaction were plated.)

    B. Convert the ng of plasmid in your transformation reaction to μg.

    C. Divide the calculated value in A by that in B.