Smash-n-Grab Plasmid Rescue from yeast

  1. Grow yeast colony in 3-4 ml SCD-Leu overnight.
  2. Transfer ~1.4 ml to a  screw-capped centrifuge tube.
  3. Centrifuge 30 sec, pour off sup.
  4. Add 200 ul plasmid rescue solution.
  5. Add 200 ul Tris-saturated (pH 8) phenol/CHCl3, vortex to mix.
  6. Add a small pea-sized amount of glass beads (Sigma G8772, 425-600 microns, acid-washed) using a clean spatula. Don't cross-contaminate.
  7. vortex vigorously 1 min. (Should form white emulsion). Repeat vortex for another minute.
  8. Spin 14,000 rpm for 5 min at RT.
  9. Remove 200 ul of top aqueous layer (= plasmid solution) to new tube (regular microfuge tube).
  10. Add 20ul 3M NaOAc salt, quick vortex to mix.
  11. Add 500 ul 95% EtOH, quick vortex to mix.
  12. Spin 10 min at 14,000 rpm, save pellet, discard sup.
  13. Add 1 ml 70% EtOH, quick vortex to mix, spin 5 minutes; carefully remove all supernatant. The pellet is very loose at this point so be careful.
  14. air-dry the plasmid pellet for 10 minutes and then resuspend in 50 ul sterile H2O.
  15. let DNA resuspend at 4 C overnight; store plasmid at -20 C.

Transforming target plasmids into bacteria

If both bait and prey plasmids carry the same antibiotic resistance gene they must be transformed into KC8 (relevant genotype leuB600, trpC9830, pyr::Tn5, hisB463, lacDeltaX74; Note: Tn5 confers kanr) to select the prey plasmid on agar plates lacking leucine because  leuB mutations can be complemented by yeast LEU2 on the prey plasmid. If bait was made using the pGBKT7 plasmid, which carries the kanR gene, the prey plasmids can be transformed into more commonly used E. coli strains, like JM109, and plated onto Amp containing plates.

  1. Mix 5 ul yeast DNA with 45 ul electro-competent E. coli cells in a chilled microcentrifuge tube on ice.
    1. Prechill electroporation cuvet on ice . (Electroporation at room temperature results in 100x lower efficiency).
    2. Pipet DNA/cells into cuvet, making sure there are no bubbles in the bottom, otherwise it will arc, ruining the transformation.
    3. Electroporate: put cuvette into slider and lock into place; push "pulse" button, a beep will sound a few seconds later after shock has been applied.
    4. Using P1000 pipettor, immediately transfer sample back into microfuge tube after shocking using 0.5 ml of LB media. Even a 1 min delay causes 3x loss of efficiency.
  2. Incubate at 37° 1 hr with shaking.
  3. Plate on minimal media plates lacking leucine for KC8 cells (amp. and kan. can be included but are optional) or LB/Amp plates; after plates have dried, incubate at 37 deg. (expect KC8 colonies after ~48 hr because leuB complementation is weak; Amp resistance colonies will show after 24 hours).
  4. Plasmid mini-prep: Grow one colony from each plate in 3-4 ml LB/amp media; after overnight growth, do a plasmid mini-prep.
  5. Digest a 10 ul of the plasmid with XhoI in 15 ul of buffer and check the size of the insert on a 1% agarose gel.. (cDNA was cloned into XhoI site of prey plasmid)
  6. Retransform original Bait yeast strain with isolated prey plasmid to confirm it encodes an interacting protein.
  7. Send off plasmid for DNA sequencing using primer FLE85 (pACT-forward primer) and FLE86 (pACT-reverse primer; optional)

     

Making electro-competent E. coli cells

 (use LB media + 50 ug/ml kanamycin for growing KC8 cells; use LB without antibiotics for other strains of E. coli, like XL-Blue)

  1. Streak E. coli strain onto an LB plate from glycerol stock and grow at 37° overnight.
  2. Innoculate 5 ml LB with a colony of bacteria, incubate at 37 deg. overnight with shaking.
  3. Next day, add overnight culture to 250 ml LB in 1 liter flask.
  4. Grow with shaking until OD600 = 0.5-0.8, which will take about 3-5 hours.
  5. Pour cells into 50 ml tubes and chill in ice/water for 20 min
  6. Spin in centrifuge at 4300 rpm for 10 min at 4° (pre-run the centrifuge to cool down).
  7. resuspend pellets in 10 ml ice-cold water and pool together
  8. spin cells at 4300 rpm 10 min at 4° .
  9. resuspend pellet in 50 ml ice-cold 10% glycerol.
  10. spin cells as in step 8 .
  11. Resuspend pellet in 1.5 ml ice-cold 10% glycerol.
  12. Aliquot 150 ul portions in sterile, labeled, chilled 1.5 ml tubes and store at -80°.