Transfer PL assemblies from Yeast to E.coli

Yeast plasmid prep:

  • Grow 3 ml of yeast culture overninght at 30oC

  • Extract plasmid from yeast using Zymoprep Yeast Plasmid Miniprep (Ref: D2001).

    1. Aliquot 1 ml of the full-grown culture in 1.5 ml microcentrifuge tubes and spin down for 2 min at 600g. Discard the supernatant

    2. Resuspend the yeast pellet in 150 μl of Solution 1

    3. Add 2 μl of Zymolase to each tube. Resuspend the pellet by flicking the tube with your finger or vortexing

    4. Incubate at 37oC for 30 min

    5. Add 150 μl Solution 2 to each tube and mix well

    6. Add 150 μl Solution 3 to each tube and mix well

    7. Centrifuge at max speed for 2min

    8. Transfer the supernatant to new tubes. Add 400 μl isopropanol to each tube and mix well

    9. Centrifuge at max speed for 8min. Aspirate the supernatant. Spin briefly againd and remove any residual supernatant

    10. Wash with 1ml of 70% fresh made ethanol

    11. Centrifuge at max speed for 8min. Aspirate the supernatant. Spin briefly againd and remove any residual supernatant

    12. Resuspend the plasmid pellet in 35 μl of H2O (not TE or EB! Since we’re going to use it for eletrophoration, and high salt concentration might decrease efficiency).

      • It’s not necessary to completely dry the pellet before resuspension.

      • Sometimes the pellet requires repeated pipetting to be completely dissolved. Use a wide bore tip to resuspend.

Electroporation of TransforMax EPI300 Electrocompetent E coli

Some considerations

  • PL plasmids will be transformed in TransforMax™ EPI300™ Electrocompetent E. coli for two main reasons:

    • They clone large inserts and transform large plasmids - up to at least 145 kb plasmid DNA.

    • They generate clones with inducible copy number using CopyControl™ vectors.

  • DNA should be in water or very low salt buffer to prevent arcing during electroporation. The pUC19 Control DNA is provided in TE at 100 pg/μl. If running a transformation control, dilute the pUC19 Control DNA 1:10 (to a final concentration of 10 pg/μl) with sterile, deionized water and use 1 μl for electroporation.

  • Prepare 1 ml of SOC or LB medium (do not include antibiotic in the medium) for each electroporation to be performed. This medium will be used for post-electroporation outgrowth of transformed cells. Maintain the medium at room temperature.

Electroporation

  1. Pre-chill electroporation cuvettes (Amaxa cuvettes work fine) and 1.5 ml tubes on ice

  2. Thaw TransforMax EPI300 Electrocompetent E. coli cells on ice. Mix gently. Use the cells immediately. Unused cells can be refrozen at –80°C.

  3. Add 2 μl of PL DNA to 20 μl of EPI300. Mix by pipetting up and down gently, If performing multiple transformations, since the elecrophoration efficiency is usually very high, each vial of competent cells can be diluted in cold water up to 3-fold to save EPI300 cells.

  4. Transfer the cell/DNA mix to the electroporation cuvette. Be sure that there are no air bubbles in the cuvette. Wipe the cuvette of any condensation. Place into the electroporator and set it into “Bacteria”. Click “Pulse” to apply the electric pulse. If the electrophoration took place correctly, the actual kV should be between 1.80 to 1.90.

  5. Immediately after electroporation, add 500 μl of room temperature SOC medium to the cuvette and transfer to a new 1.5ml tube

  6. Incubate at 30°C with shaking at 220-230 rpm for 1 hour to recover the cells and allow expression of the antibiotic resistance marker.

  7. Plate each transformation in two LB plates (90% and 10%) on appropriate antibiotic (Kan for LM1110-based assemblies). For cells transformed with the control pUC19 DNA, plate on LB agar containing 100 μg/ml of ampicillin. The remaining cell outgrowth can be stored at 4°C in the event additional cell dilutions are plated.

  8. Incubate the plates at 30°C overnight