Plasmid Prepping

To use plasmids for TC work, they must be purified by prepping on one of three scales: mini, midi, or maxi.

Miniprep

This protocol is adapted from the NEB protocol (provided in the box). Minipreps yield ~40 uL of DNA at ~100 ng/uL.

  1. Under a flame, aliquot 3 mL of media matching the plasmid antibiotic resistance to culture tubes.

  2. Pick a single colony or dab a glycerol stock with a toothpick or pipette tip.

  3. Let the bacteria grow overnight, no longer than 24 hrs.

Note

Grow viral plasmids in 30C to reduce the chance of recombination. Non-viral plasmids may be grown at 37C for better yield.

  1. Spin down bacteria at 4800g for 5 minutes in centrifuge by shakers.

  2. Pour of supernatant in cells + media waste.

Warning

Do not pour media into miniprep waste or vice versa! If this occurs, add bleach in the fume hood and allow fumes to disperse. Talk to EHS rep.

  1. Resuspend pellet in B1 (kept in deli fridge) and transfer to new 1.7 mL tube.

  2. Add 200 uL B2 and gently invert 5-6 times to lyse cells. Incubate at RT for 1 minute.

  3. Add 400 uL B3 and invert until uniformly yellow (no pink). Incubate for 2 minutes.

Note

MC adds 250 uL of B2 and immediately after addition starts a time for 90 seconds, inverts for 30 seconds, and adds 500 uL of B3 at 90 seconds.

  1. Spin down on benchtop centrifuge at max speed for 5 minutes.

  2. Transfer lysate to filter column placed on a fresh 1.7 mL tube. Discard pellet.

  3. Spin lysate at max speed for 1 minute.

  4. Aspirate lysate in miniprep waste.

  5. Add 200 uL of Wash 1 (Buffer BZ).

  6. Spin at max speed for 1 minute.

  7. Add 400 uL of Wash 2 (Buffer WZ).

  8. Aspirate wash in miniprep waste.

  9. Dry spin at max speed for 1 minutes.

  10. Transfer column to fresh 1.7 mL tube.

  11. Add 40 uL of Elga water and incubate for at least 1 minute.

  12. Spin at max speed for 1 minute to elute DNA.

  13. To quantify yield, use 2 uL of sample for nanodrop reading.

Tip

Pre-warm Elga water in 55C bath for better elution.

Midiprep

This protocol is adapted from the Qiagen protocol (provided in the box). Midipreps yield ~200 uL of DNA at ~500 ng/uL.

  1. Under a flame, aliquot 50 mL of media matching the plasmid antibiotic resistance to a 250 uL flask.

  2. Pick a single colony or dab a glycerol stock with a toothpick or pipette tip.

  3. Let the bacteria grow for ~24 hrs.

  4. Harvest bacteria by transferring to 50 mL conical and centrifuging at 6000g for 15 minutes at 4C.

Note

The fixed conical rotor is in the cabinet below the other rotors.

  1. Pour out supernatant in cells + media waste.

  2. Resuspend pellet in 4 mL of Buffer P1 (kept in deli fridge).

  3. Add 4 mL of Buffer P2 and gently invert 5-6 times to lyse cells. Incubate at RT for 3 minutes. The suspension should turn blue.

  4. While you wait, place filter column on a new 50 mL conical tube.

  5. Add 4 mL Buffer S3 and mix 4-6 times, or until the lysate is completely colorless.

  6. Transfer lysate to filter column placed on the 50 mL conical tube and incubate for 10 min.

  7. During incubation, place the small spin columns onto the vacuum manifold and add tube extenders.

  8. Gently insert plunger into filter column to filter lysate.

  9. Add 2 mL Buffer BB to cleared lysate and invert 4-6 times.

  10. Connect manifold to vacuum pump from miniprep waste, and turn on until all liquid passes through.

Note

Commonly, not all liquid will be able to pass through. To overcome this, you can add 700 uL at a time of lysate to the spin column and centrifuge at max speed for 1 min, or directly put the spin column onto the vacuum tube.

  1. Add 700 uL Buffer ETR and apply vacuum until all liquid has been drawn through all columns.

  2. Add 700 uL Buffer PE and apply vacuum until all liquid has been drawn through all columns.

  3. Dry spin at max speed for 1 minutes to remove residual wash buffer.

  4. Place spin column onto fresh 1.7 mL tube.

  5. Add 200 uL of Buffer EB and incubate for at least 2 minutes.

  6. Spin at max speed for 1 minute to elute DNA.

  7. To quantify yield, use 2 uL of sample for nanodrop reading.

  8. Store plasmids at -20C.

Maxiprep

This protocol is adapted from the Qiagen protocol (provided in the box). Maxipreps yield ~400 uL of DNA at ~500 ug/uL. This protocol is essentially identical to midipreps, just with larger volumes. We typically only maxiprep the packaging and envelope plasmids for viral production, which require sequencing after every prep given the likelihood of recombination.

  1. Under a flame, aliquot 100 mL of media matching the plasmid antibiotic resistance to a 500 mL flask.

  2. Pick a single colony or dab a glycerol stock with a toothpick or pipette tip.

  3. Let the bacteria grow for ~24 hrs.

  4. Harvest bacteria by transferring to 2x50mL conicals and centrifuging at 6000g for 15 minutes at 4C.

Note

The fixed conical rotor is in the cabinet below the other rotors.

  1. Pour out supernatant in cells + media waste.

  2. Resuspend pellet in 8 mL of Buffer P1 (kept in deli fridge). You can add 4 mL to each conical and add them together after resuspending separately.

  3. Add 8 mL of Buffer P2 and gently invert 5-6 times to lyse cells. Incubate at RT for 3 minutes. The suspension should turn blue.

  4. While you wait, place filter column on a new 50 mL conical tube.

  5. Add 8 mL Buffer S3 and mix 4-6 times, or until the lysate is completely colorless.

  6. Transfer lysate to filter column placed on the 50 mL conical tube and incubate for 10 min.

  7. During incubation, place the small spin columns onto the vacuum manifold and add tube extenders.

  8. Gently insert plunger into filter column to filter lysate.

  9. Add 5 mL Buffer BB to cleared lysate and invert 4-6 times.

  10. Connect manifold to vacuum pump from miniprep waste, and turn on until all liquid passes through.

Note

Commonly, not all liquid will be able to pass through. To overcome this, you can add 700 uL at a time of lysate to the spin column and centrifuge at max speed for 1 min, or directly put the spin column onto the vacuum tube.

  1. Add 0.7 mL Buffer ETR and apply vacuum until all liquid has been drawn through all columns.

  2. Add 0.7 mL Buffer PE and apply vacuum until all liquid has been drawn through all columns.

  3. Dry spin at max speed for 1 minutes to remove residual wash buffer.

  4. Place spin column onto fresh 1.7 mL tube.

  5. Add 400 uL of Buffer EB and incubate for at least 2 minutes.

  6. Spin at max speed for 1 minute to elute DNA.

  7. To quantify yield, use 2 uL of sample for nanodrop reading.

  8. If prepping packaging and/or envelope plasmids, normalize concentration to 500 ng/uL and send a sample for sequencing.

  9. Store plasmids at -20C.