The alkaline lysis method is used to prepare plasmids from Escherichia coli. It is a routine technique used every day in molecular biology research laboratories. But I have been in graduate school for more than ten years. Almost without exception, those who feel like I know everything are excellent. The students know very little about the principle of alkaline plasmid extraction. I think it is probably because molecular clones only talk about the experimental steps, but do not elaborate on the principles. This is the main reason that led my students to go astray Afterwards, I found out that the level of graduate students in related fields in China is almost the same, and even many teachers are in this state. This has to be sad.

I think this may have something to do with our culture. Chinese people adore reading. The concept of learning and optimizing is deeply rooted in people's hearts. Parents often say to their only seedlings that you only need to concentrate on reading a good book. So the definition of reading It is to remember the things in the textbooks and get high scores during the exam. This is the fundamental reason why modern science has not sprouted in China. If Chinese culture does not change at this point, then science cannot really take root in China. It can only be transformed into a new eight-legged life science, which is experimental science. It focuses on hands-on. If experimental science only needs to read a book, then I would like to ask that fairy who would read a book and ride a bicycle? Or listen to the physical education teacher's explanation and you will skate? But if you don't think about it, you will become a craftsman? A qualified life science researcher needs to perfect himself in these two aspects. An outstanding scientific worker is an artist who is familiar with scientific principles and good at application. Every classmate who has extracted plasmids by alkali method, I hope you will read this article Can think about something and give China's future a hope

For ease of understanding, here are three solutions used for alkaline plasmid extraction:
Solution I, 50 mM glucose / 25 mM Tris-Cl / 10 mM EDTA, pH 8.0;
Solution II, 0.2 N NaOH / 1% SDS;
Solution III, 3 M potassium acetate / 2 M acetic acid

Let ’s take a look at the role of solution I in any biochemical reaction. First, we must control the pH of the solution. Therefore, it is natural to use a Tris-Cl solution with an appropriate concentration and an appropriate pH value. So what does 50 mM glucose do? What about? Unbelievably speaking, the biggest benefit after adding glucose is that the suspended E. coli will not quickly deposit to the bottom of the tube. Therefore, if glucose is missing in solution I, it will have almost no effect on the extraction of the plasmid itself. So say Glucose is missing in solution I. What about EDTA? As we all know, EDTA is a chelating agent for divalent metal ions such as Ca2 + and Mg2 +. The main functions of EDTA in molecular biology reagents are: to inhibit the activity of DNase and to inhibit the growth of microorganisms. Adding up to 10 mM EDTA to solution I is nothing more than To chelate all the divalent metal ions in E. coli cells. If EDTA is not added, it ’s actually not a big deal. As long as you do n’t do foreign work, as long as you complete the plasmid extraction in a very long time, you do n’t have to worry about DNA. It is rapidly degraded because there is EDTA in the TE buffer that finally dissolves the plasmid. If one day you just lack solution I, can you extract the plasmid? To tell you the truth, just use equal volume of water, or LB medium to suspend the bacteria. One thing that should not be forgotten is that the bacteria must be suspended evenly, and there must be no lumps.

It's turn for solution II. This is mixed with equal volume of fresh 0.4 N NaOH and 2% SDS. It should be diluted with concentrated NaOH to prepare 0.4 N NaOH. It is nothing more than to ensure that NaOH does not absorb CO2 in the air and weaken. Many people do not know that in fact, the main reason for breaking cells is alkali, not SDS, so it is called alkaline extraction. In fact, NaOH is the best reagent for lysing cells. Whether it is E. coli or mammalian cells, alkali is encountered Will dissolve almost instantly, this is due to the phase change of the cell membrane from the bilayer structure to the microelle structure, which results in the use of stale 0.4 N NaOH, even with SDS, it cannot be effectively dissolved Escherichia coli (may try it yourself), naturally it is difficult to extract plasmids with high efficiency. If only SDS can be used, of course, a small amount of plasmids can also be extracted, because SDS is also alkaline, but it is weak. Many people have the effect on NaOH. Misunderstood is to denature genomic DNA in order to precipitate, this is due to not correctly understanding the description of the denaturation of DNA in some books, some people can not help but ask, since it is NaOH solution Solution cells, why do you need to add SDS? That is a stepping stone for the next operation. There are two points to remember in this step: First, the time should not be too long. Never answer the phone at this time, because under such alkaline conditions, the genomic DNA fragments will slowly break; Second, it must be mixed gently (like treating girls), otherwise genomic DNA will also break. Breaking of genomic DNA will cause trouble, I will explain in detail later

Everyone knows that there will be a large amount of precipitation after adding solution III, but most people do not understand the nature of this precipitation. The most misunderstood is that the precipitation that occurs when SDS encounters acidity. If you are so doubtful, go to 1 Add 2M acetic acid solution to the% SDS solution to see if it is not the case. A large amount of precipitation appears, which is obviously related to the addition of SDS. What will happen if SDS is not added in solution II, there will also be a small amount of precipitation However, the amount is much less. Obviously the protein precipitated by salting out and acid denaturation. Since SDS is not a precipitation due to acid, will it be a precipitation due to salt? Slowly add 5 N NaCl to the 1% SDS solution. You will find that SDS will precipitate at high salt concentration. Therefore, high concentration of salt causes the precipitation of SDS. But if you add not NaCl but KCl, You will find that the amount of precipitation is much more. This is actually sodium dodecylsulfate (potassium dodecylsulfate, PDS), which is insoluble in water. Therefore, precipitation occurs. It seems that the precipitation after the addition of solution III is actually potassium ions replacing sodium ions in SDS to form insoluble PDS, and the high concentration of salt makes the precipitation more complete. Everyone knows that SDS specifically likes protein Combined, an SDS molecule is bound to an average of two amino acids. The large amount of precipitation produced by potassium and sodium ion replacement naturally precipitates most of the protein. It is pleasing that the genomic DNA of E. coli is also co-precipitated together. This process does not It's hard to imagine, because the genomic DNA is too long, the long DNA is naturally easy to co-precipitate by PDS, although SDS does not bind to DNA molecules

So why is 2 M acetic acid added? It is to neutralize NaOH, because long-term alkaline conditions will break the DNA, so once the genomic DNA to be neutralized breaks, as long as it is a fragment of 50-100 kb in size, there is no way to co-precipitate it by PDS so the alkali The processing time should be short, and it should not be vigorously shaken, otherwise there will always be a lot of genomic DNA mixed in the resulting plasmid. Agarose electrophoresis can observe a thick total DNA band. Many people mistakenly think that the solution III is added to the genome. DNA can be precipitated without rapid renaturation, which is a big misunderstanding, because denatured or renatured, DNA molecules are dissolved in neutral solution. NaOH was originally used to dissolve cells. DNA The denaturation of the molecule is actually a by-product, and it does not matter whether it is precipitated or not. Solution III is added and mixed evenly and placed on ice, the purpose is to precipitate PDS more fully

Don't think that the formation of PDS precipitate can precipitate all the proteins. In fact, there are still many proteins that cannot be precipitated. Therefore, extraction with phenol / chloroform / isoamyl alcohol, and then alcohol precipitation to obtain stable quality plasmid DNA, Otherwise, it will degrade due to the mixed DNase in a long time. There are many reasons to use 25/24/1 phenol / chloroform / isoamyl alcohol. Here is a comprehensive introduction to the degeneration of phenol (Phenol) on protein. In chloroform, phenol should be used to extract the protein to the greatest extent, but the specific gravity of water-saturated phenol is slightly heavier than water, and it encounters a high concentration of salt solution (such as 4M guanidine isothiocyanate). After centrifugation, the phenol phase It will go to the upper layer, which is not conducive to the recovery of the aqueous phase containing the plasmid; but after adding chloroform, the specific gravity can be increased, so that the phenol / chloroform is always in the lower layer, which is convenient for the recovery of the aqueous phase; there is another point, phenol and water are very miscible If a large amount of phenol is dissolved in the aqueous phase after extraction with phenol alone, phenol will inhibit many enzyme reactions (such as restriction enzyme digestion reaction), so if you use phenol extraction alone, you must use chloroform for extraction The phenol in the water phase is removed once, and the phenol / chloroform mixture is used for extraction. The phenol running into the water phase is much less. Trace amounts of phenol will be removed during ethanol precipitation without worrying about enzymes The reaction such as cutting cannot proceed normally. As for the addition of isoamyl alcohol, its function is mainly to make the interface between the upper and lower layers clear after centrifugation, and also facilitate the recovery of the aqueous phase

The recovered water phase contains enough salt, so as long as 2 volumes of ethanol are added, the plasmid DNA can be precipitated by centrifugation after being left at room temperature for a few minutes. The precipitation is different from the ordinary DNA alcohol precipitation recovery, so do n’t be too careful. High concentration of salt will hydrate a large number of water molecules, so it is easy to form hydrogen bonds between the DNA molecules and precipitate. If you feel that salt precipitation occurs , Just wash it with 70% ethanol several times, each time at room temperature for more than one hour, and use the tip to break the precipitate, you can get a good sample. The plasmid sample is generally used with RNase (50 ug / ml) TE Buffer to dissolve, otherwise a large amount of undegraded RNA will interfere with the electrophoresis results

When agarose electrophoresis is used to identify plasmid DNA, in most cases you can see three bands, but do not think that you are seeing super-helix linear and open-loop three-band alkaline extraction to obtain plasmid samples without linear DNA If you do n’t believe it, you can use EcoRI to linearize the plasmid and then perform agarose electrophoresis. You will see that the position of the linear plasmid DNA is different from the position of the three bands. In fact, the three bands are sorted according to the speed of the electrophoresis speed. Ring opening and replication intermediates (that is, two plasmids that are not completely replicated are connected together) If you accidentally shake excessively after adding solution II, there will be a fourth band, this band swims slowly, away from this The three bands are fragments of E. coli genomic DNA of 20-100 kb. Very occasionally, sometimes the extracted plasmids will have 7-10 bands. This is due to the special DNA sequence leading to different degrees of supercoil (super The number of turns of the spiral is different)

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