ProtocolsLithiumAcetateTransformation < Lab

---+ 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 &lt;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
Barrick Lab > ProtocolList > ProtocolsLithiumAcetateTransformation
Topic revision: r1 - 2017-02-23 - 21:16:11 - Main.DaciaLeon