---+ Lithium Acetate Transformation This protocol is originally from the [[http://www.cshl.edu/][Cold Spring Harbor Laboratory]] Yeast Genetics & Genomics course manual. ---++ Making Competent _S. cerevisiae_ Cells This procedure makes enough competent cells for 5-10 transformations. 1 Grow an overnight culture of each strain in YPD or selective media. * The culture should be grown in flasks instead of tubes so the cells are properly aerated. 1 Measure OD600: * If OD600 is greater than or equal to 1.0, dilute cells back to 0.1 and grow for 4-6 hours. * If OD600 is 0.2-1.0, use immediately or dilute for use later in the day. * If OD600 is <0.2, continue growing cells. 1 Transfer the cells to 50 ml conical tubes 1 Pellet cells by centrifugation for 3-5 minutes at 2500 RPM. 1 Remove media and resuspend the cell pellet in 10 ml of 1x LiOAc buffer by vortexing. 1 Centrifuge the cells for 3 minutes at 2500 RPM. 1 Remove liquid and resuspend pellet in 0.5-1.0 ml of 1x LiOAc buffer. 1 Divide into 100 µl aliquots in Eppendorf tubes. 1 Proceed to transformation. ---++ Transforming _S. cerevisiae_ Cells with Lithium Acetate 1 Prepare single stranded carrier DNA. * Thaw salmon sperm carrier DNA on ice, boil for 3 minutes, then cool on ice. 1 Combine 100 µl of competent cells in 1x LiOAc buffer with: *10 µl of 10 mg/ml carrier DNA *1-5 µg of plasmid DNA or 25% of an efficient PCR (maximum 10 µl of total volume). 1 Add 280 µl of PEG solution to each tube. * Vortex or pipette vigorously to mix the PEG with other components. 1 Incubate 20-45 minutes at room temperature. 1 Add 43 µl of DMSO to each tube. Vortex or pipette to mix thoroughly. 1 Incubate 5-15 minutes at 42°C. 1 Immediately chill on ice for 3 minutes. 1 Centrifuge cells for 2 minutes at half-speed (1250 RPM) and remove liquid. 1 Add 0.5 ml of sterile water, vortex briefly, spin down, and remove liquid. 1 For auxotrophic selection, resuspend in 200 µl of TE buffer. For antifungal, resuspend in 200 µl of YPD. 1 Spread cells on appropriate selective plates and incubate for 2-3 days. ---+++ Notes 1 This protocol yields ~500-5000 colonies/µg of plasmid DNA. Consider plating high and low volumes of cell suspension (e.g. 160 µl and 40 µl) to obtain plates with well-spaced transformants. 1 You do not need to boil the carrier DNA every time. Keep a small aliquot in a -20°C freezer and re-boil after three or four freeze/thaw cycles. 1 In our experience, incubating the cells with PEG for 45 minutes leads to a higher transformation efficiency. 1 If cells are incubated at 42 °C for longer than 15 minutes, the cells may go into heat shock resulting in a failed transformation. ---++ Materials 1x LiOAc buffer * 0.1 M LiOAc * 10 mM Tris-HCl (pH 8.0) * X mM EDTA 1 M LiOAc 1 Dissolve 51 g of LiOAc in 450 ml of water. 1 Adjust volume to 500 ml. 1 Filter sterilize or autoclave. 10x TE * 10 ml 0.5 M EDTA * 50 ml 1 M Tris HCl (pH 8.0) * 440 ml sterile ddH2O Single-stranded carrier DNA * 10 mg/ml salmon sperm DNA * 10 mM Tris-HCl (pH 8.0) * 1 mM EDTA Boil for 3 minutes, cool on ice, and store at -20°C in aliquots. PEG solution 1 Heat 50 ml ddH2O in microwave. 1 Add: * 50 g PEG 3350 * 10 ml 10x TE * 10 ml 1 M LiOAc 1 Adjust the volume to 100 ml. 1 Filter sterilize with a 0.45 µm filter unit. 1 Store PEG solution in tightly capped container. -- Main.DaciaLeon - 23 Feb 2017
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Topic revision: r1 - 2017-02-23 - DaciaLeon