Plasmid Isolation Using Alkaline Lysis

Plasmid Isolation Protocol

1. 5 ml LB medium containing proper antibiotics were inoculated with a single bacterial colony. The tube was incubated at 37 ˚C overnight with vigorous shaking at 360 rpm.
2. Pellet bacteria from the culture at 10,000 x g for 5 minutes at room temperature.
3. Discard the supernatant.
4. Resuspend bacterial pellet in a total of 1 ml ice-cooled solution I (50 mM). Pipet up and down or vortex as necessary to fully resuspend the bacteria.
5. Add 2 ml room temperature 0.2 N NaOH/1.0% SDS to the suspension. Mix thoroughly by repeated gentle inversion. Do not vortex.
6. Add 1.5 ml ice-cold Solution III to the lysate. Mix thoroughly by repeated gentle inversion. Do not vortex.
7. Centrifuge at 15,500 x g for 30 minutes at 4C.
8. Recover resulting supernatant.
9. Add 2.5 volume isopropanol to precipitate the plasmid DNA. Mix thoroughly by repeated gentle inversion. Do not vortex.
10. Centrifuge at 15,500 x g for 30 minutes at 4C.
11. Removal of resulting supernatant. The pellet is plasmid DNA.
12. Rinse the pellet in ice-cold 70% EtOH and air-dry for about 10 minutes to allow the EtOH to evaporate.
13. Add ddH2O or TE to dissolve the pellet. After the addition of 2ul RNase A (10mg/ml), the mixture was incubated for 20 minutes at room temperature to remove RNA. 

Note:

• Spin down your cells. Your DNA is still in the cells, so it is in the pellet at this stage.
• Discard the supernatant and to even invert the tube and wipe the lip with a Kim-wipe or Q-tip.
• Resuspend the cells in buffer (often Tris) and EDTA. EDTA chelates divalent metals (primarily magnesium and calcium). Removal of these cations destabilizes the cell membrane. It also inhibits DNases. Glucose should also be added to maintain osmolarity and prevent the buffer from bursting the cells. 
• Lyse the cells with sodium hydroxide (NaOH) and SDS. This highly alkaline solution gave rise to the name of this technique. Mix this by gentle inversion and incubate on ice for five minutes (but no longer, or your DNA will be irreversibly denatured). 
• Three things happen during this stage: 

1. SDS pops holes in the cell membranes. SDS (sodium dodecyl (lauryl) sulfate) is a detergent found in many common items such as soap, shampoo, and toothpaste.
2. NaOH loosens the cell walls and releases the plasmid DNA and sheared cellular DNA.
3. NaOH denatures the DNA. Cellular DNA becomes linearized and the strands are separated. Plasmid DNA is circular and remains topologically constrained.

• Renature the plasmid DNA and get rid of the garbage. Add potassium acetate (KAc), which does three things:

1. Circular DNA is allowed to renature. Sheared cellular DNA remains denatured as single-stranded DNA (ssDNA).
2. The ssDNA is precipitated since large ssDNA molecules are insoluble in high salt.
3. Adding sodium acetate to the SDS forms KDS, which is insoluble. This will allow for the easy removal of the SDS from your plasmid DNA. 

Now that you've made it easy to separate many of the contaminants, centrifuge to remove cell debris, KDS, and cellular ssDNA. Your plasmid DNA is in the supernatant, while all of the garbage is in the pellet.

• Precipitate the plasmid DNA by alcohol precipitation (ethanol or isopropanol) and a salt (such as ammonium acetate, lithium chloride, sodium chloride or sodium acetate) and spin this down. DNA is negatively charged, so adding a salt mask the charges and allows DNA to precipitate. This will place your DNA in the pellet.
• Rinse the pellet—your plasmid DNA—in ice-cold 70% EtOH and air-dry for about 10 minutes to allow the EtOH to evaporate.
• Resuspend your now clean DNA pellet in the buffer (often Tris) and EDTA plus RNases to cleave any remaining RNA. Your DNA is now back in solution. 

DNA of this purity is good for a number of uses, such as in vitro transcription or translation or cutting with some enzymes. If you are sequencing or transforming this DNA into mammalian cells, you'll want to use additional purification techniques such as phenol extraction, Qiagen column purification, or silica-based purification.