Notebook

Prepare Agar Plates with Nutrient Agar

  1. Place agar bottles in a water bath at 90°C until the agar liquifies.
  2. Leave at room temperature to cool down to approximately 50°C so it can be handled.
  3. Pour 10 mL of the liquified agar onto 5 cm petri dishes and leave them at room temperature to cool down.
  4. Transfer the plates to the refrigerator till the agar solidifies (overnight).

Inoculation with E. coli

  1. Place agar plates and bacterial stab inside the incubator at 30°C.
  2. Once warmed, transfer them to the hood to start the inoculation.
  3. Scrape the culture from the bacterial stab with a wire loop and inoculate each plate (negative control).
  4. Poured 50 μL of ampicillin sensitive E. coli and swab onto the plates (positive control).
  5. Place 3 ampicillin disks on each plate once the inoculation was complete.
  6. Transfer the plates into the incubator and let them grow overnight.

Plate Quality Control

  1. Once your plates have solidified and dried, test them to make sure the antibiotic functions properly.
  2. Take out two plates.
  3. On the first plate, streak out a strain that you know to be resistant to the antibiotic.
  4. On the second plate, streak out a strain that’s not resistant to the antibiotic.
  5. Incubate both plates overnight at the appropriate growth temperature and check for growth.
Plate quality control

Bacterial Inoculation

  1. Using a sterile pipette tip or toothpick, select a single colony from your agar plate.
  2. Drop the tip or toothpick into the bottle with the nutrient liquid and swirl.
  3. Loosely unscrew the cap.
  4. Incubate bacterial culture at 30°C overnight in the incubator.
  5. After incubation, check for growth, which is characterized by a cloudy haze in the media.
Bacterial growth 1 Bacterial growth 2 Bacterial growth 3

Maxiprep

Buffer Preparation

  1. Add 88 ml of 95-100% ethanol to the 23 ml ZymoPURE™ Wash 2 (Concentrate) before use.
  2. If ZymoPURE™ P2 or Binding Buffer has precipitated, dissolve by incubating at 30-37°C for 10-20 minutes and mix by inversion. Do not microwave.

Before Starting

Centrifuge up to 150 ml of bacterial culture at ≥ 4.4 RPM for 25 minutes to pellet the cells (wet pellet ~5 ml). Discard supernatant.

Plasmid DNA Purification

  1. Add 4 ml of cold ZymoPURE™ P1 (Red) to the bacterial cell pellet and resuspend completely by vortexing or pipetting.
P1 buffer resuspension 1 P1 buffer resuspension 2
  1. Add 4 ml of ZymoPURE™ P2 (Blue) and gently invert 6 times. Let sit 2-3 min at room temperature. Cells are lysed when solution appears clear, purple, and viscous.
P2 lysis
  1. Add 4 ml of ZymoPURE™ P3 (Yellow) and invert gently 5 more times after it turns completely yellow. A yellowish precipitate will form.
P3 neutralization
  1. Attach plug to Luer Lock at bottom of ZymoPURE™ Syringe Filter-X. Place the syringe upright and load lysate. Wait 5-8 minutes for precipitate to float.
  2. Remove Luer Lock plug and push solution through filter until ~10 ml cleared lysate is recovered. Save cleared lysate.
Syringe filter 1 Syringe filter 2
  1. Remove and discard 15 ml Reservoir-X from Zymo-Spin™ V-PX Column. Centrifuge 13.5 RPM for 1 min to remove residual wash buffer.
Centrifuge column

Elution & Storage

  1. Transfer column into clean 1.5 ml tube, add 400 μl ZymoPURE™ Elution Buffer, incubate 2 min, centrifuge ≥16,000 × g for 1 min.
  2. Optional: Remove endotoxins with EndoZero™ Spin-Column.
  3. Dilute eluate 1:10 before spectrophotometer reading.
  4. Store eluted plasmid DNA at ≤ -20°C.

Plasmid Transformation Confirmation - Agarose Gel Electrophoresis

  1. Weight 0.5 g agarose powder.
  2. Mix with 50 ml 1x TAE buffer in a microwavable flask.
Agarose in TAE
  1. Microwave for 1 min until agarose dissolves.
  2. Cool to ~50°C.
  3. Pour agarose into gel tray with comb in place.
  4. Add 3 μL Midori Green dye to agarose solution.
Add dye
  1. Let gel solidify (4°C 10-15 min or room temp 20-30 min).
Add TAE over gel
  1. Add loading buffer to DNA samples (4 μL per 15 μL) and pipette ~5 μg DNA into wells.
Add loading buffer
  1. Place gel into electrophoresis box, cover with 1x TAE or TBE.
  2. Load molecular weight ladder in first lane.
  3. Load samples into remaining wells.
  4. Run at 80-150 V until dye is 75-80% down (~1–1.5 h).
Running gel
  1. Visualize DNA fragments using a fluorescence imaging device.
Visualize gel

Note: The experiment was not continued due to insufficient yeast culture density for transformation.