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Plasmid Purification from E. coli

We used two plasmid miniprep protocols. The first protocol is taken from the GenElute Miniprep Plasmid Preparation Kits distributed by Sigma Aldrich. We modified the elution portion of the procedure by using double distilled water to elute rather than using TE buffer. We also skipped the step with the optional wash solution. Instead, the step with the addition of Wash Solution in the Column Tube was done twice.

  1. Make overnight cultures from LB agar plate growth (The protocol for the making of overnight cultures can be found as a separate protocol).
  2. After allowing approximately 16 hours of growth, pellet the cells using a centrifuge for 20 minutes at a speed of 4000 rpm at 4°C.
  3. Discard the supernatant, while being careful not to discard any of the pellet.
  4. Resuspend the pellet in 200 µL of Resuspension Solution (with RNase A added) provided from the kit. In case of Rhodococcus plasmid purification, 20 µL of lysozyme with a concentration of 20mg/mL was added and the tube was incubated at 37°C for 30 minutes.
  5. Transfer the solution from a Falcon tube to a 1.5 µL microcentrifuge tube.
  6. Add 200 & µL of Lysis Solution and invert gently to mix. Allow the mixture to clear for less than 5 minutes.
  7. Add 350 & µL of Neutralization Solution and invert the tube 4-6 times to mix.
  8. Pellet the microcentrifuge tubes at 14000 rpm using a microcentrifuge for 10 minutes. The resulting solution is the lysate.
  9. Add 500 µL of the Column Preparation Solution to a binding column inside a collection tube. Centrifuge this tube for 1 minute at 14 000 rpm and discard the liquid underneath the binding tube.
  10. Transfer the lysate into the binding column, being careful not to transfer any solid. Discard the microcentrifuge tube with the solid.
  11. Centrifuge the collection tube at 14 000 rpm for 1 minute. Discard whatever liquid flowed through the binding column into the collection tube.
  12. Add 750 µL of Wash Solution with concentrated ethanol added to the column and spin at 14 000 rpm for 1 minute. Discard the liquid that flowed through into the collection tube.
  13. Repeat Step 12 a second time with the same quantity of Wash Solution.
  14. Centrifuge the tube for 1 minute at 14 000 rpm to dry the column.
  15. Transfer the column to a new 1.5 mL microcentrifuge tube.
  16. Add 50 µL of double distilled water to the column and spin for 1 minute at 14 000 rpm.
  17. Use a spectrophotometer to measure the concentration and the purity of your plasmid.

The second protocol uses in-house reagents and ethanol precipitation of plasmid DNA. The required buffer solutions are:

  • P1 : 50 mM TrisHCl (pH 8.0), 10 mM EDTA, 100 µg/ml RNAse A (store at 4°C)
  • P2 : 200 mM NaOH, 1% SDS
  • P3 : 3 M KAc (pH 5.5) (store at 4°C)
  1. Grow 2.5 mL O/N culture and use 2 mL for below, keeping 0.5 mL for glycerol stock.
  2. Pellet culture into 2 mL microfuge tube.
  3. Aspire supernatant; Repeat if necessary.
  4. Resuspend pellet in 300 µL P1 (Keep on ice).
  5. *Perform next quickly (<1 min). * Add 300 µL P2 → Invert → Add 300 µL P3
  6. Centrifuge 14000 rpm for 10 min @ RT.
  7. Aliquot the supernatant into 1.5 mL microfuge tube (~600-800 µL).
  8. Add 650 µL isopropanol (RT) → Invert → Incubate for 10 min at room temperature.
  9. Centrifuge 14000 rpm for 10 min @ 4°C → Aspirate.
  10. Wash pellet with 70% cold (-20°C) EtOH (~500 µL).
  11. Centrifuge 14000 rpm for 5 min @ 4°C → Aspirate.
  12. Air-dry pellet (place tubes upside down or SpeedVac for 10 min).
  13. Resuspend (by flicking) in double distilled water (30 µL).
  14. Leave standing at RT for a few minutes to facilitate dissolving of plasmid in double distilled water.
  15. Run 3-4 µL on gel to check quality AND/OR Measure concentration (A260/A280).