Yeast Transformation

Updated 10/9/2020 02:06 PM

Step-by-step

by Franka Butzbach (created by Philip Schulz)

Introduction

Now the fun part begins

Materials

YPD or the appropriate SD liquid medium

Sterile 1XTE/1X LiAc (Prepare immediately prior to use from 10X stocks; stock recipes in Appendix D.B)

Sterile 1.5-ml microcentrifuge tubes for the transformation

Appropriate SD agar plates (100-mm diameter)

Appropriate plasmid DNA in solution (check amounts required)

Appropriate yeast reporter strain for making competent cells (check volume of competent cells required; Steps 1–11 of Section V.E will give you 1.5mL, enough for 14–15 small-scale transformations)

Carrier DNA (Appendix D.B) • Sterile PEG/LiAc solution (Prepare only the volume needed, immediately priorto use, from 10X stocks; Appendix D.B)

100% DMSO (Dimethyl sulfoxide; Sigma Cat No. D-8779)

Sterile 1XTE buffer (Prepare from 10XTE buffer; Appendix D.B)

Sterile glass rod, bent Pasteur pipette, or 5-mm glass beads for spreading cells on plates.

Procedure

Prepare yeast one day before transformation

Inoculate 1mL of YPD or SD (synthetic dropout) with several colonies, 2–3 mm in diameter.

Note: For host strains previously transformed with another autonomously replicating plasmid, use the appropriate SD selection medium to maintain the plasmid (Appendix E).

Vortex vigorously for 5 min to disperse any clumps.

Transfer this into a flask containing 50mL of YPD or the appropriate SD medium.

Note: take a sterile erlenmeyer flask as there needs to be room for oxygen

Incubate at 30°C for 16–18 hr with shaking at 250 rpm to stationary phase (OD600>1.5).

Transformation

Work sterile: always open flasks etc. near a lit Bunsen-Burner!

Transfer 15mL of overnight culture to a flask containing 300mL of YPD. Check the OD600 of the diluted culture and, if necessary, add more of the overnight culture to bring the OD600 up to 0.2–0.3

Note: alternitively we can use 5mL of the ON and inoculate 100mL of YPD (just make sure that there is a 1:20 ratio of ON:YPD)

Incubate at 30°C for 3 hr with shaking (230 rpm). At this point, the OD600 should be 0.4–0.6. Note: If the OD600 is <0.4, something is wrong with the culture (see Troubleshooting Section F.6).

Keep the competent yeast cells on ice!!

Thaw Salmon Sperm (DNA carrier). Once thawed, cook it at 92°C for 10min (heating block)

Place cells in 50-ml tubes and centrifuge at 1,000 x g for 5 min at room temperature (20–21°C).

Discard the supernatants and thoroughly resuspend the cell pellets in sterile TE or distilled H₂O. Pool the cells into one tube (final volume 25–50mL).

Centrifuge at 1,000 x g for 5 min at room temperature.

Decant the supernatant.

Resuspend the cell pellet in 1.5mL of freshly prepared, sterile 1X TE/1X LiAc.

Note: our TE and LiAc are 10X. We therefore need to dilute them 1:10 and then mix the two components 1:1 (check with supervisor if that's actually true)

Add 0.1µg of plasmid DNA and 0.1mg of carrier DNA to a fresh 1.5-ml tube and mix.

Notes:

Use 1μg of plasmid! -> need to calclater how much ul! (see benchling entry from 25.08 and multiply those volumes by 10)

Add 10μl of carrier (salmon sperm) to 1.5mL tube

For simultaneous cotransformation (using two different plasmids), use 0.1µg of each plasmid (an approximately equal molar ratio), in addition to the 0.1mg of carrier DNA.

For transformations to integrate a reporter vector, use at least 1µg of linearized plasmid DNA in addition to the carrier DNA.

Add 0.1mL/100µL of yeast competent cells (cells in 1.5mL 1:10 TE/LiAC) to each tube and mix well by vortexing.

Add 0.6mL of sterile PEG/LiAc solution to each tube and vortex at high speed for 10 sec to mix.

10mL PEG/LiAc solution:

8mL of 50% PEG (viscous, pour it directly)

1mL of 10X TE

1mL of 10X LiAc

Incubate at 30°C for 30 min with shaking at 200 rpm.

Preheat waterbath to 42°C (DON'T PUT ANYTHING INTO THE WATER YET!!!)

Add 70µL of DMSO. Mix well by gentle inversion. Do not vortex.

DMSO can be found below the fumehood in P₁-38. (Dimethylsulfoxid bottle with red cap)

pipette the needed amount into a 1.5mL tube.

Heat shock for 15 min in a 42°C water bath.

In the mean time, prepare the 1X TE buffer needed in step 22. Make a 1:10 dilution (eg. 1mL TE, 9mL sterile water).

Chill cell s on ice for 1–2 min.

Centrifuge cells for 5 sec at 14,000 rpm at room temperature. Remove the supernatant.

Use table centrifuge: set to 13.3 for 1min and stop centrifuge at around 50-45seconds. -> peletts will form and supernatant becomes "clear" and easy to remove.

Resuspend cells in 100µL of sterile 1X TE buffer.

Plate 100μl on Yeast dropout media -TRP plate

From script: Plate 100µL on each SD agar plate that will select for the desired transformants. To ensure that you will obtain a plate with well-separated colonies, also spread 100µL of a 1:1000, 1:100, and 1:10 dilution on 100-mm SD agar plates. These will also provide controls for (co)transformation efficiency.

Note: If you are performing a cotransformation, plate controls to check transformation efficiency and markers of each plasmid. On separate 100-mm plates, spread 1µL (diluted in 100µL H₂O) on medium that will select for a single type of plasmid.

Incubate plates, up-side-down, at 30°C until colonies appear (generally, 2–4 days).

Say some kind words & give the plates a kiss for extra fast colony growth and superior results :D