Purification of Plasmid DNA

(Miniprep aka. Ethanol Lysis)

Protocol:

Day 0: Grow chosen bacteria + plasmid on a fresh LB-Agar-Antibiotic Plate

  1. In order to maximise the growth of your bacteria in the liquid media, ensure you have a relatively fresh (<2 weeks) plate of bacteria to inoculate from.

    • If you only have plasmid material, do the heat shock protocol today and then inoculate from your heat shock plate tomorrow.

    • If you have plates that are older than 2 weeks, re-streak them onto a fresh plate and inoculate from this plate tomorrow.

Day 1: Inoculation

  1. Using the plasmid map as a guide, add 5-15 µl of the correct antibiotic diluted to the stock concentration to your 5-15 ml of sterile LB media.

    • If you have a shaking incubator, 5 ml will be more than sufficient. If your incubator doesn't shake or you don't have an incubator at all, you can increase the volume to 15 ml to compensate. If your antibiotic stock solution is at the correct concentration then;
      5 ml Media = 5 µl antibiotic
      15 ml Media = 15 µl antibiotic

  2. Set up a sterile workspace.

  3. Flame your inoculating loop and use sterile technique to transfer a single colony into the liquid media.

  4. Leave the lid loose enough for air transfer, then incubate at 37 degrees with shaking 8-16 hrs or overnight.

Day 2: Miniprep

  1. Pellet cells in a 15 ml Falcon tube by centrifuging at max speed for 5 minutes.
    If you only have a small centrifuge, grow your cells in 5 ml then use 3-4 microcentrifuge tubes, trying to to get all the cells pelleted in the minimum number of tubes.

    • Label your tubes carefully to ensure you don't pellet different plasmids together.

    • Always be extremely careful to balance your centrifuge before spinning.

  2. Pour off the supernatant(s)

  3. Resuspend pelleted bacterial cells in 250 μl Buffer P1 Resuspension Buffer by vortexing or by using your micropipette to disturb the cell pellet. Transfer all of the suspension to a microcentrifuge tube.
    Return the tube of P1 to the fridge as soon as you're done - it contains RNAse which will slowly denature at RT.

  4. Equip yourself with a lab-coat and gloves if you haven't already and consider wearing goggles. The next few buffers range from mild to extremely toxic, consult the buffer mixing guides to learn more.

  5. Add 250 μL Buffer P2 Lysis Buffer to each tube and invert gently 8-10 times. Incubate the tubes on the bench for 10 minutes.

    • Note; no vigorous shaking from here onward, you will shear your plasmid DNA.

  6. Add 350 μL Buffer N3 Neutralisation Buffer to each tube and invert gently 8-10 times.
    You should see a thick precipitate form, white if you are not using indicator. If you are using indicator dye, keep inverting until all traces of blue disappear.

  7. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.

    • Always be extremely careful to balance your centrifuge before spinning.

    • If your centrifuge can't go this fast the pellet will not be very compact, but you can overcome this with careful pipetting in the next step.

  8. While you wait, assemble your silica spin columns and capture tubes. Label them carefully to match their corresponding tubes (currently spinning in the centrifuge). Also label a fresh set of 1.5 ml centrifuge tubes with the same label - these will be the tubes we eventually elute our DNA into for storage in the freezer.
    Once the spin finishes, line all the tubes up in your tray to minimise the risk of pipetting errors.

  9. Carefully remove the supernatant without disturbing the white pellet of cell debris and decant it into the top of the silica spin column.

    • You can throw out the tubes containing the white precipitate, all your plasmid DNA is in the supernatant.

  10. Centrifuge the silica spin columns in their capture tubes for 60 s at max speed.

    • If you notice some of the liquid hasn't passed through the silica then your max speed may be a bit low. Compensate by spinning for longer.

  11. Optional: You can repeat this plasmid capture step step to improve yield. .Open the centrifuge and one-by-one remove the silica column + capture tube, pick up the flow-through in the capture tube with your pipette and add it back into the top of the spin column. Don't worry about it falling through the silica and onto the bench, it won't. Respin the tube 60s, max speed, to push the plasmid DNA through the silica filter a second time, potentially doubling the amount captured.

  12. Discard the flow through into your miniprep waste container, it should not go with other liquid waste. Remember to follow proper disposal protocols.

    • Your plasmid DNA should now be bound to the silica column.

  13. Wash the silica spin column by adding 500 μl Buffer PB Wash Buffer 1 and centrifuging for 60 s. Discard the flow-through into miniprep waste.

  14. Wash the silica spin column by adding 750 μl Buffer PE Wash Buffer 2 and centrifuging for 60 s. Discard the flow-through into miniprep waste.

  15. Centrifuge silica columns at full speed for an additional 1 min to remove residual wash buffer.

  16. Remove silica columns from their capture tubes and place on a kimwipe and into 50°C Oven for 15-30 minutes

    • Alternatively place the columns into fresh capture tubes and leave in a heat block at 60°C for 15-30 minutes

  17. Place the silica spin column in the clean 1.5 ml microcentrifuge tube that you labelled earlier. To elute DNA, add 30-50 μl TE/Water/EB Elution Buffer to the center of each silica spin column, let stand for 1 min, and centrifuge at max speed for 1 min.

    • Keep an eye on the lids of the tubes when you place them in the centrifuge since they will be hanging loose. If they're going to slap into the walls of the centrifuge, snip them off with a pair of scissors and label a fresh set of tubes to move your elution into after the centrifuge step.

    • Note: Less volume of Elution Buffer will result in a higher concentration of plasmid DNA.

    • If you’re noticing significant volume loss at this stage, consider increasing the length or speed of the spin.

  18. Optional: As with the capture spin, you can repeat this elution step with the same volume of liquid to potentially increase your yield. Using a micropipette, extract the eluted Plasmid DNA and place it back onto the spin column , let it stand for 1 min, then centrifuge at max speed for 1 min.

    • This trick is also useful if you had to split the same plasmid between multiple spin columns and now need to elute into a single tube, without reducing the final concentration. Simply Babushka-doll the eluted plasmid and buffer into each additional column, using the same 30-50μl of EB one at a time for each column.

  19. Dispose of the spin column, then fasten the lid on your microcentrifuge tube. Double check the label before storing at -20°C.

    • A really clean purification of Plasmid DNA in TE that is stored at -20°C can last for more than a decade.

    • The EDTA in TE will inhibit any enzymatic reactions however, so consider using EB or Water if you plan on using any enzymatic reactions downstream. The purified plasmid DNA will still last multiple years before degrading.

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