Plasmid Isolation Protocol

Procedure of Plasmid Isolation

1. Harvest Bacterial and Resuspended Cells

* Choose a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate for approximately 8 h at 37°C with vigorous shaking (approx. 300 rpm).

* Dilute the starter culture 1/500 to 1/1000 into 3 ml selective LB medium. Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm).

* Harvest the bacterial cells by centrifugation at 6000 x g for 15 min and remove as much of the supernatant as possible. Resuspend the bacterial pellet in 0.1-0.5 ml of resuspension buffer (50mM Tris-Cl, 10 mM EDTA, 100 µg/ ml RNase A, pH 8.0). The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

2. Cell Lysis

* Add 0.25 ml of lysis buffer, mix thoroughly by vigorously inverting the sealed tube 4–6 times, and incubate at room temperature (15–25°C) for 5 min. Do not vortex, as this will result in shearing of genomic DNA. The lysate should appear viscous. Do not allow the lysis reaction to proceed for more than 5 min.

3. Neutralization

* Add 0.3 ml of neutralization buffer, mix immediately and thoroughly by vigorously inverting 4–6 times, and incubate on ice for 5 min. Precipitation is enhanced by using chilled neutralization buffer and incubating on ice. After addition of neutralization buffer, a fluffy white material forms and the lysate becomes less viscous. The precipitated material contains genomic DNA, proteins, cell debris, and KDS. The lysate should be mixed thoroughly to ensure even potassium dodecyl sulphate precipitation. If the mixture still appears viscous, more mixing is required to completely neutralize the solution. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.

4. Load Lysate on Column

* Before loading the column, carefully remove the supernatant and then transfer it to a collection tube containing the column and centrifuge at 13,000 rpm for 1 minute.

* Discard the flow-through liquid and remove supernatant containing plasmid DNA promptly. After centrifugation, the supernatants should be clear.

* If the supernatant is not clear, a second, shorter centrifugation should be carried out to avoid applying any suspended or particulate material to the column. Suspended material (which causes the sample to appear turbid) will clog the column and reduce or eliminate flow.

5. Bind and Wash

* Add 0.7 ml of wash buffer to the column placed in the collection tube and centrifuge for 10 minutes at 13000 rpm for 1 minute. Equilibrate by applying 1 ml equilibration buffer ( 750 mM NaCl, 50 Mm MOPS, ph 7.0, 15 % isopropanol ) and allow the column to empty by gravity flow. Flow of buffer will begin automatically by reduction in surface tension due to the presence of detergent in the equilibration buffer.

* Apply the supernatant from step 6 to the column and allow it to enter the resin by gravity flow.

6. Plasmid Elution

* Elute DNA with 0.8 ml elution buffer (1.23 M NacL, 50 mm Tris-Cl, pH 8.5, 15 %v isopropanol) Collect the elute in a 1.5 ml or 2 ml microcentrifuge tube.

* Precipitate DNA by adding 0.7 volumes (0.56 ml per 0.8 ml of elution volume) of room-temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥10,000 rpm for 30 min in a microcentrifuge. Carefully decant the supernatant. All solutions should be at room temperature in order to minimize salt precipitation.

* Wash DNA pellet with 1 ml of 70% ethanol and centrifuge at 10,000 rpm for 10 min.

* Carefully decant the supernatant without disturbing the pellet.

* The 70% ethanol removes precipitated salt and replaces isopropanol with the more volatile ethanol, making the DNA easier to redissolve.

* Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of buffer (e.g., TE buffer, pH 8.0, or 10mM Tris-Cl, pH 8.5). Redissolve the DNA pellet by rinsing the walls to recover all the DNA.

7. Determination of yield

To determine the yield, DNA concentration should be determined by both UV spectrophotometry at 260 nm and quantitative analysis on an agarose gel. To quantitate the nucleic acid concentration, dilute the plasmid DNA 1 : 100 or 1 : 50 (depending on the plasmid copy number) in TE buffer and measure the absorbance (optical density) at 260 nm (A260) and 280 nm (A280). Use TE buffer as the blank. This measurement permits the direct calculation of the nucleic acid concentration using the formula.

[DNA] (μg/mL) = A260 × Dilution factor × 50

where 50 is the extinction coefficient of DNA. This measurement method can directly calculate the nucleic acid concentration using formula.