Plasmid extraction reagents are the most basic and simple experiments in molecular biology. There is almost no need to use the brain during the experiment, we just need to directly follow the instructions in the extraction kit. Although the operation process is simple, plasmid extraction is also a relatively important step. The quality and quantity of the proposed plasmid will directly determine the success or failure of the subsequent laboratory. When following the instructions in the kit, we will see different solutions (different kits may be marked differently). So do you know what is in each bottle of solution? What is their role in the plasmid extraction process? Plasmid extraction is very simple, but the knowledge behind it is not simple.

　　Plasmid extraction adopts strong alkaline lysis method (alkaline lysis), which was invented by Birnboim and Doly in 1979, and the citations of the original article on SCI have reached 12886 so far. So let's now look at what the solutions P1, P2, N3, PE, and EB are.

　　Solution 1 (P1)

　　Component concentrations 25 mM Tris-HCl (pH 8.0), 10 mM EDTA, 50 mM Glucose

　　The main function of solution 1 is to suspend the bacterial cell pellet. 25mM Tris-HCl is a buffer solution to keep the pH of the reaction system constant. EDTA is a metal ion chelator. The function of 10 mM EDTA is to combine with metal ions in microorganisms and inhibit the activity of DNase. The function of 50 mM glucose is to increase the viscosity of the solution, ensure the suspension of the bacteria, and delay the time for the sedimentation of the bacteria. RNase (RNase A) is usually added to solution 1 before use. The role of RNase is very clear, degrading RNA in the solution. Since RNase is a protein with poor protein stability, solution 1 with RNase A needs to be stored at 4oC at low temperature.

　　Solution 2 (P2)

　　Component concentration 250 mM NaOH, 1% (W/V) SDS (sodium dodecyl sulfate)

　　The main effect of solution 2 is cell lysis. After solution 1 suspends the cells, it is necessary to add solution 2. The function of solution 2 is to lyse the cells. Solution 2 includes only two components: sodium hydroxide and SDS. What really plays the role of cell lysis is sodium hydroxide, which is why this method is called alkaline lysis method. Sodium hydroxide will damage the structure of the cell membrane, causing it to undergo a phase change from the bilayer (bilayer membrane) structure to the micelle (microcapsule) structure, resulting in cell lysis. The role of SDS will be mentioned in the next step. One thing to pay attention to after adding solution 2 is that the time must not be too long, and the other is that the centrifuge tube should not be shaken violently. Excessive time and severe vibration will cause sodium hydroxide to damage the genomic DNA, and the fragmented genomic DNA fragments will be extracted together with plasmids of similar size, contaminating the sample.

　　Solution 3 (N3)

　　Component concentration 3M potassium acetate (potassium acetate), 5M acetic acid

　　The main function of solution 3 is to neutralize the strong base added in the second step and remove impurities such as proteins in cells. N is an abbreviation for neutralized. The strong alkaline solution sodium hydroxide will destroy its structure with DNA base for a long time, so it needs to be neutralized. Neutralizing sodium hydroxide, 5 M acetic acid is enough, why add potassium acetate? Students who have done plasmid extraction experiments should remember that after adding solution N3, there will be a lot of precipitation. These precipitates actually interact with the SDS in solution 2 to produce potassium dodecylsulfate (PDS). After the addition of solution N3, SDS encounters potassium ions, resulting in PDS that is insoluble in water and precipitates rapidly.

　　So how does the addition of solution 3 remove impurities such as protein and genomic DNA? The reason is that sodium dodecyl sulfate (SDS) added in solution 2 is easily combined with proteins, and an average of two amino acids will combine with one SDS molecules, the two combine to form the SDS2 polypeptide complex, so that the denatured protein will dissolve in solution, allowing the polypeptide chain to better entangle with the genomic DNA. After adding solution 3, since the dodecyl sulfate combined with the denatured protein into a complex, the precipitation of potassium dodecyl sulfate precipitated the denatured protein together, and at the same time the denatured protein was entangled with the genomic DNA, and the result was mostly co-precipitated. The solution with high ion concentration accelerates the precipitation process. In addition, the surface charge of the denatured protein is reduced after the solution is neutralized, which can also promote the precipitation of the denatured protein.

　　Solution PE (wash buffer)

　　Component concentrations 10 mM Tris-HCl (pH 7.5), 80 % Ethanol

　　The main function of the Wash buffer is to wash away excess salt ions. All kits use silica gel columns for DNA extraction. After the DNA is adsorbed on the silica column, the excess salt ions need to be washed off with Wash buffer. The Wash buffer contains a high concentration of ethanol. Since ethanol will affect subsequent enzyme digestion or sequencing reactions, the column must be "emptied" in a centrifuge before DNA elution to completely remove the ethanol.

　　Solution EB (Elution buffer)

　　Component concentration 10 mM Tris-HCl (pH 7.5)

　　The role of EB is to elute the DNA sample on the silica column. Elution buffer should not contain too high concentration of salt ions, because in a high-salt environment, salt cations will break the hydrogen bond between the silica negative oxygen radical and water, so that the whole silica gel carries positive charges, which will attract negatively charged DNA molecules . In addition, heated eluent (<50°C) can accelerate DNA detachment from silica membrane. In addition, keeping the EB buffer on the membrane for a longer time before centrifugation will be more conducive to the elution of DNA.

　　The composition of the solution in the plasmid extraction kits of different companies may be different. For example, glucose can be removed in P1, and the solution PE can even be directly replaced with water. The plasmid extraction experiment is a basic and important experiment in molecular biology. Although the experiment itself is simple, its principle is still somewhat complicated. The editor believes that if you know the reason, you must also know the reason, and if you know the principle of the experiment, when there is a problem in the experiment, it will be easier to find the reason and correct it.