When extracting plasmids by alkali lysis, what is the reason why the buffer added first suspends the

I don't know, I'm just looking.

The main components in solution 2 are NAOH and SDS, which are used to lyse cells. Because Naoh encounters carbon dioxide.

It is easy to react to form calcium bicarbonate.

It affects the use effect, so it should be used now. In the case of SDS, precipitation is likely to occur at low temperatures. However, a water bath at 37°C is fine. The main problem is NAOH.

Solution 1 is mainly to let the cell cells fully suspend in the solution, which has no practical significance, and can be omitted.

Solution 2 is lye with the aim of completely destroying the cell membrane.

Release nucleic acids. Solution 3 is an acidic potassium salt.

The solution is dissolved, the purpose is to neutralize the alkalinity and precipitate the SDS, along with the protein and the large linear DNA.

After adding the solution of extracting plasmid DNA by alkali lysis method, one solution, two solutions, and three solutions, their phenomena are different, and it can be seen that the reason for the obvious difference is that the process of their reaction is different, and the chemical reaction is also different.

Glucose is so that the suspended E. coli does not quickly deposit to the bottom of the tube; EDTA is a chelating agent of divalent metal ions such as Ca2+ and Mg2+, and its main purpose is to chelate divalent metal ions to achieve DNase inhibition activity. RNase A can be added to digest RNA

Specifically, these phenomena should be decomposition phenomena, and the cause should be some chemical changes.

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The addition of solution one solution and two solutions to extract plasmid DNA by alkali lysis method will produce many phenomena after three.

Answer] :(1) The main components of the solution are glucose and EDTA. Glucose can increase the viscosity of the solution and prevent the degradation of residual DNA by mechanical action. EDTA chelates Mg2+

Ca2+ and other metal ions inhibit the degradation of DNA by DNase (DNase requires certain metal ions as cofactors).

2) The main component of the solution is sodium hydroxide (NaOH) and is stable in the solution of pH 5 9, but when pH 12 or pH 3, it will cause the dissociation and denaturation of hydrogen bonds between DNA duplexes. The concentration of NaOH in the solution is that, after adding the solution, the pH of the system is , thus promoting the denaturation of chromosomal DNA and plasmid into single strands. SDS is an anionic detergent, and its main functions are:

dissolves fats and proteins on the cell membrane, thereby dissolving membrane proteins and destroying the cell membrane; Depolymerization of nuclear proteins in the cell, separating the proteins from the DNA; SDS can bind to proteins to form SDS-protein complexes, which denature and precipitate proteins. SDS inhibits the action of nucleases and prevents degradation of DNA.

3) The main component of the solution is KAC (. The KAC solution used is to adjust the pH of the solution to neutral, so that the denatured plasmid DNA can be refolded and can be stably presented. The high-salt 3mol L potassium acetate (KAC) is conducive to the condensation and precipitation of denatured macromolecular chromosomal DNA, RNA and SDS-protein complexes.

In general, if you experience degradation of plasmid DNA after extraction using alkaline lysis, there are a few potential solutions you can try. First, you should make sure that you are using the correct concentration of base for your specific plasmid and that you carefully follow the extraction protocol. If you are unsure of the correct concentration or protocol, you should refer to the protocol provided by the manufacturer or refer to the protocol guidelines for your specific plasmid.

Second, you can try using different extraction methods. There are many different methods for plasmid DNA extraction, each with its own advantages and disadvantages. Some common alternatives to alkaline hydrolysis include mechanical disruption, enzymatic digestion, and ion-exchange chromatography.

Third, you can try different methods to purify plasmid DNA after extraction. There are many purification methods available, each with its own advantages and disadvantages. For example, you can try to purify plasmid DNA using ethanol precipitation, column group purification, or gel electrophoresis.

In conclusion, the key to solving this problem is to carefully evaluate your protocol and try different methods until you find the right one for your specific plasmid and extraction conditions. It may also be helpful to consult with other researchers or knowledgeable lab technicians for advice and guidance.