Maxiprep of plasmid DNA from E. coli

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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 acetic acid -- glacial is 17M)
• 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.

Notes

You may want to read this discussion of what LiCl and sodium acetate are doing in this protocol.

BioStream version

Following is the Maxiprep of plasmid DNA from E.coli protocol in BioStream, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioStream (see Source code). More information about BioStream can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi

Text Output

Maxiprep of plasmid DNA from E.coli protocol

Source Code

Maxiprep of plasmid DNA from E.coli protocol - source code