Maxiprep of plasmid DNA from E. coli

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(Ingredients)
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* 111μL 20mg/mL lysozyme
* 111μL 20mg/mL lysozyme
* 2mL worth of components for solution 2: 200μL 10% SDS, 100μL 4M NaOH, 1.7 mL autoclaved water
* 2mL worth of components for solution 2: 200μL 10% SDS, 100μL 4M NaOH, 1.7 mL autoclaved water
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* 1.5mL Solution 3: 3M K<sup>+</sup>, 5M acetate
+
* 1.5mL Solution 3: 3M K<sup>+</sup>, 5M acetate (3M potassium-acetate, 2M glacial acetic acid)
* 3.7mL isopropanol
* 3.7mL isopropanol
* 1mL TE buffer
* 1mL TE buffer

Revision as of 19:06, 17 March 2007

This protocol produces very clean, concentrated DNA, usually on the order of several mg/mL. It is labor intensive. Expect to spend most of the day doing it, but I like to store my plasmids in this form.

Ingredients

Ingredients are per culture; make enough for one extra culture to allow for pipetting error).

  • 150μL sterile 50% glycerol
  • 1mL TEG (25mM Tris-Cl, 10mM EDTA, 50mM dextrose)
  • 111μL 20mg/mL lysozyme
  • 2mL worth of components for solution 2: 200μL 10% SDS, 100μL 4M NaOH, 1.7 mL autoclaved water
  • 1.5mL Solution 3: 3M K+, 5M acetate (3M potassium-acetate, 2M glacial acetic acid)
  • 3.7mL isopropanol
  • 1mL TE buffer
  • 0.5mL 5M LiCl
  • 7.5μL 1mg/mL RNaseA
  • 200μL 70% ethanol
  • several mL of phenol:chloroform:isoamyl alcohol (25:24:1)
  • several mL of chloroform:isoamyl alcohol (24:1)
  • 750μL straight ethanol
  • 125μL 3M sodium acetate

Directions

  1. Grow a single colony of E. coli overnight in 50mL LB broth + selective markers at 37°C.
  2. The next morning, put 850μL of the culture in each of two Eppendorf tubes, add 150μL sterile 50% glycerol, and store at -80°C. Pour as much culture as will fit into an Oak Ridge tube and centrifuge at 5800g/6000rpm, 4°C, for 10 minutes in a GSA rotor. Discard the supernatant, add the rest of the culture, and repeat. Resuspend in 1mL TEG.
  3. Add 111μL 20mg/mL lysozyme. Incubate on ice for 30 minutes. Meanwhile, mix: 250μL 10% SDS, 125μL 4M NaOH, 2.125mL autoclaved water per culture.
  4. Add 2mL SDS/NaOH mix to each tube. Incubate on ice for 10 minutes.
  5. Add 1.5mL Solution 3 (3M K+, 5M acetate). Incubate on ice for 10 minutes.
  6. Shake vigorously. Centrifuge in SS34 rotor at 17,200g/12,000rpm, 4°C, for 15 minutes.
  7. Pour the supernatant into another Oak Ridge tube and discard the pellet. Add 2.7mL isopropanol. Centrifuge at 17,200g/12,000rpm (room temperature) for 10 minutes. Discard the supernatant.
  8. Wash pellet with 1mL 70% ethanol. Air dry for 2-5 minutes on bench. Resuspend in 500μL TE buffer. Add 500μL 5M LiCl. Incubate on ice for 5 minutes.
  9. Centrifuge at 17,200g/12,000rpm for 10 minutes.
  10. Pour supernatant into an Eppendorf. Add 1mL isopropanol. Incubate on the bench for 10 minutes.
  11. Centrifuge at 17,200g/12,000rpm for 10 minutes.
  12. Discard the supernatant. Wash the pellets with 100μL 70% ethanol. Resuspend in 375μL TE buffer. Add 7.5μL 1mg/mL RNaseA. Incubate at 37°C for 30 minutes.
  13. Add 700μL phenol:chloroform:isoamyl alcohol. Vortex until thoroughly mixed. Centrifuge at top speed of microfuge for 2 minutes. Pipette aqueous phase (the top one) into new Eppendorf. Repeat until the interface between the phases is clear after centrifugation. Then repeat the procedure twice with chloroform:isoamyl alcohol to remove any phenol.
  14. Add 750μL straight ethanol and 125μL 3M sodium acetate. Put at -80°C for 30 minutes or -20°C overnight.
  15. Centrifuge at 13,600g/12,000rpm, 4°C, for 15 minutes. Discard the supernatant. Wash pellet with ~100μL 70% ethanol. Resuspend in 100-200μL TE buffer.
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