Streamlined Plasmid DNA Purification

Isolation of high-quality plasmid DNA from the gemisch of the E. coli bacterial lysate is essential for many research laboratory as well as clinical genetic
applications. Most of the plasmids in common use today replicate so profusely that sufficient DNA is readily available from a small-scale preparation (mini-prep) to
accommodate many of the routine tasks. Clearly, plasmid DNA isolation in the genomic era calls for a method with features of speed, simplicity, high quality, and
low cost.

Our mini-prep kit is based on a modified and streamlined alkaline lysis method (see below), further eliminating the needs for additional ribonuclease treatment and
phenol-chloroform extraction. Plasmid DNA can be rapidly purified from an overnight E. coli culture in 15 min with a typical yield of 1 – 5 ug DNA per ml of culture
(as dictated by plasmid replication origin). DNA to be used for sequencing analysis is subjected to an additional 10-min step of PEG precipitation. Up to 1,000
bases can typically be resolved per DNA sequencing run. Our Maxi-Plasmid Isolation Kit can purify larger quantity of DNA in 90 min with a typical yield of 0.5 – 3
ug DNA per ml of culture. Purified DNA typically has an A260/A280 ratio of ~2.

Alkaline lysis is the method of choice for isolating closed circular plasmid DNA from bacteria. This method capitalizes on the facts that plasmid DNA, unlike the
chromosomal DNA, is able to rapidly anneal following alkaline denaturation and that potassium dedecyl sulfate, compared to its sodium counterpart, has a much
lower water solubility. Several key steps used in the alkaline lysis mini-prep protocol are outlined and principles explained.

1. Chelation. The bacterial cell pellet is resuspended in Solution-1 containing EDTA, which chelates divalent metals such magnesium and calcium. Removal of
these cations through EDTA chelation destabilizes the cellular structure and inhibits nuclease activity.

2. Alkalization. Cells are then gently ruptured by Solution-2 containing SDS (sodium dodecyl sulfate) and NaOH. SDS is a detergent found in many household
items such as soap, shampoo and toothpaste. It pops holes in the cell membranes. NaOH loosens the cell walls, shears as well as denatures the chromosomal
DNA, and releases the plasmid DNA. Plasmid DNA is circular, and remains topologically constrained.

3. Neutralization. The pH of the lysate is neutralized by adding Solution-3 containing acetic acid and potassium acetate. This step causes plasmid DNA, but not
cellular DNA, to renature rapidly and remain soluble. Most of the chromosomal DNA and bacterial proteins form massive precipitates along with the potassium
dodecyl sulfate precipitate.

4. Garbage Disposal. Now we can simply remove many of the contaminants and unwanted debris by centrifugation. The plasmid DNA is in the supernatant, while
most of the garbage is in the pellet.

5. DNA Harvesting. The supernatant contains plasmid DNA and some cell wall components (polysaccharides). Although the DNA phosphate backbone is
negatively charged, the potassium and sodium ions already abundantly present in the solution carry positive charges and thus shield the negative charges of
DNA. Adding an alcohol at this point triggers DNA aggregation through non-ionic hydrophobic interaction. This will place the plasmid DNA in the pellet after
another centrifugation. Pelleted DNA is finally brought up in water, and is ready for use in many laboratory applications.

Remember you will be moving your plasmid DNA from the cell to the supernatant and to the pellet during the isolation procedure. The last thing you need is to
accidentally dump the supernatant or pellet when that is where your DNA is. OK. It’s 11PM already. Do you know where your plasmid DNA is?
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