Wittrup: Yeast Transformation
From OpenWetWareJump to navigationJump to search
For making libraries using electroporation:
- Inoculate an EBY100 colony (freshly streaked on a YPD plate) in 5 mL YPD and grow overnight at 30 °C.
- Inoculate a 50 mL culture of YPD to OD600=0.1 using overnight culture from Step 1 and grow cells at 30 °C until OD600 ~1.3-1.5 (about 6 h).
- Cells must be in early to mid-log growth phase. Use of cells in late log or stationary phase decreases transformation efficiency >10-fold.
- While cells are growing, precipitate DNA for transformation using PelletPaint according to the manufacturer’s protocol. Typically, four electroporation cuvettes, each with 5 ug insert and 1 ug backbone, are used to generate a library of ~5x10^7. Also prepare a backbone-only control. Leave DNA in pellet form.
- Insert-to-backbone ratios can be varied from 5:1 to 1:1 with at least 1 ug backbone per cuvette.
- Once cells have reached the appropriate OD600, add 500 μL of freshly prepared Tris/DTT buffer (1 M Tris pH 8.0, 2.5 M 1,4-dithiothreitol, filter-sterilized) to the culture. Incubate in shaker at 30 °C for 15 min.
- Transformation efficiency is relatively constant for DTT incubation times of 10-20 min, but decreases considerably for incubation over 20 min.
- Pellet cells and wash with 25 mL E buffer (10 mM Tris pH 7.5, 270 mM sucrose, 1 mM MgCl2, filter-sterilized) (rinse, pellet, and aspirate supernatant). Wash cells again with 1 mL E buffer. Perform washes at 4 °C and with ice-cold E buffer. Make sure to remove all supernatant, as contaminants carried over can cause decreased transformation efficiency.
- Resuspend cells to 300 uL total volume in E buffer. Resuspend DNA and control pellets using the appropriate volume of cell suspension (50 μL/cuvette). Keep cells on ice.
- Aliquot 50 μL resuspended cell-DNA mixture per pre-chilled electroporation cuvette (2 mm gap). Keep electroporation cuvettes on ice until pulsed.
- Load cuvette into gene pulser and electroporate at 0.54 kV, 25 uF without a pulse controller. Immediately add 1 mL warm (30 °C) YPD to the cuvette.
- Typical time constants for electroporation range from ~15-40 ms without significantly impacting transformation efficiency (~1000Ω)
- Transfer cells from pulsed cuvettes to a 15 mL Falcon tube. Wash out remaining cells in cuvette with an additional 1 mL of YPD.
- Shake cells for 1 h at 30 °C.
- Pellet cells and remove supernatant. Resuspend in 10 mL SDCAA. Plate serial dilutions on SDCAA plates to determine transformation efficiency. The backbone only control should have an efficiency of less than 1% of the backbone plus insert transformations.
- Transfer cell suspension to a flask with 100-1000 mL SDCAA + 1:100 pen-strep solution. Incubate at 30 °C for 24-48 h.
- Passage library at least once before use to reduce number of untransformed cells.
Alternate library method: lithium acetate transformation (Geitz protocol):