AQUAPLASMID

Simple. One-Solution. Inexpensive. Plasmid Miniprep.

AquaPlasmid™ is a multifunctional reagent for plasmid DNA purification without using columns. This single solution completes cell suspension, cell lysis, plasmid DNA extraction and cell debris removal in a single tube. AquaPlasmid is nontoxic. The multifunctionality makes AquaPlasmid the most economic ($0.66/miniprep) and environment-friendly (nontoxic) plasmid purification product on the market. The isolated plasmid DNA is pure and suitable for all downstream applications, including automated DNA sequencing.

AquaPlasmid Brief Protocol

(Download AquaPlasmid User Manual for plasmid miniprep, midiprep, and maxiprep protocols)

1. Harvest the Cells

Transfer 1.5 ml of overnight bacterial culture to a 1.7-ml microfuge tube. Centrifuge at 14,000 xg for 1 min at 22 °C to pellet the bacteria. Aspirate or flip the tube forcefully a few times to remove the culture medium as completely as possible.

2. Lyse the Cells

Add 200 ul of AquaPlasmid solution to the cell pellet. Immediately vortex for 10-20 sec to fully suspend the cells. Incubate at 22 °C for 5 min to lyse the cells. After the incubation, touch-vortex (1-2 sec on and 1-2 sec off) at top speed twice (IMPORTANT: Do not over-vortex or it could cause genomic DNA contamination).

3. Remove the Debris

Incubate the crude lysate at -20 °C for 5 min or on ice for 10 min to induce precipitation. Centrifuge at 14,000 xg for 5 min at 22 °C to pellet the cell debris (centrifuge for 10 min at 4 °C may produce tighter debris pellet).

4. Pellet the Plasmid DNA

Transfer the clear lysate (~200 ul) to a clean 0.5-ml microfuge tube. Add 0.5 vol (~100 ul) of isopropanol to the lysate (do not use more than 0.7 vol of isopropanol or it may increase small RNA contamination). Touch-vortex at top-speed 5-10 times to mix well. Centrifuge at 14,000 xg for 5 min at 22 °C to pellet the plasmid DNA. Flip the tube (you may hold 6-10 tubes between your thumb and index finger at the same time) forcefully a few times to discard the supernatant.

5. Rinse the Plasmid DNA

To rinse the DNA pellet, gently fill up the tube and its lid with 70% ethanol from a squirt bottle and then flip the tube to discard the ethanol solution. Repeat the 70% ethanol rinse once. Flip the tube forcefully a few times and tap it on a paper towel to remove residual ethanol. Let the DNA pellet air-dry for 5-10 min. Add 50 ul of deionized water (or TE buffer) to the DNA pellet. Vortex to fully suspend the DNA pellet and incubate at 22 °C for 5 min. Centrifuge at 14,000 xg for 2 min to pellet any insoluble. Optional: Transfer the DNA solution to a new tube.

FAQ about AquaPlasmid

1. Do I need to store the AquaPlasmid kit at 4 °C?

No, AquaPlasmid solution should be stored at room temperature (~22 °C). If the temperature is below 18 °C, precipitates may develop. If so, incubate the solution at 37-55 °C for a few minutes and vortex to re-solubilize it before use.

2. How does AquaPlasmid work?

AquaPlasmid combines the functions of traditional P1 buffer (cell suspension), P2 buffer (cell lysis), N3 buffer (debris removal), and silica column (plasmid DNA purification) in a single solution. It lyses the cells without denaturing the DNA, extracts the plasmid DNA while keeping other cellular contaminants in the cell debris. The plasmid DNA is subsequently precipitated from the clear lysate with isopropanol.

3. Does AquaPlasmid contain guanidine salts?

No, AquaPlasmid does not contain guanidine salts that are required for column-based purification. Guanidine salts are not biodegradable. When mixed with Bleach, they can release toxic and mutagenic fumes into the environment, which may be harmful to lab workers and aquatic lives.

4. How should I scale up and down the reagent for other culture volumes?

We recommend using 200 ul of AquaPlasmid for each 1.5 ml of overnight bacterial culture. However, if the cell density is too high, it may result in poor cell lysis, incomplete debris removal and decreased DNA yield. An indication of cell density exceeding the capacity of AquaPlasmid is a large DNA pellet following isopropanol precipitation. In that case, you should use 300 ul of AquaPlasmid for each 1.5 ml of bacterial culture or use 200 ul of AquaPlasmid for each ml of culture.

5. Can I use AquaPlasmid to extract plasmid DNA from endA1 strains?

We recommend AquaPlasmid only for plasmid extraction from E. coli strains carrying the endA1 mutation, such as TOP10, DH5a, XL-1 Blue, JM109, and SURE, etc. If the genotype is unknown, you should incubate an aliquot of the purified DNA in a restriction enzyme buffer at 37 °C for 12 hours to confirm there is no DNA degradation. If the DNA is degraded, you may try incubating the purified DNA at 75-85 °C for 30 min to inactivate the contaminating nucleases.