The principle of the function of bio-chemical reagents commonly used in molecular experiments is summarized.

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1. Lysolysase: It is glycoside hydrolyzed enzyme, which hydrolyzed the β-1,4 glycoside bonds in the main chemical component of the cell wall of the bacteria, and therefore has the role of lysate. When pH in the solution is less than 8, the lysolysase action is inhibited. Glucose: Increases the viscosity of the solution, maintains osmosis pressure, and prevents
DNA
degradation by mechanical shear forces.. EDTA
:(1) chelates metal ions such as Mg2 plus and Ca2 plus, inhibits the degradation of DNA by DNA (DNase requires certain metal ions as a co-base) ;(2) EDTA presence, which is conducive to the role of lysase, because the reaction of lysolyses requires a lower ion strength environment.
2. NaOH-
SDS
: NaOH: Nucleic acids are stable in solutions with pH greater than 5 and less than 9. But when pH > 12 or pH<3, it causes the hydrogen bond between the double strands to dissocess and denature. The NaOH concentration in Solution II is 0.2mo1/L, and when pumped, the pH of the system is as high as 12.6, which causes the degeneration of chromosomal DNA and proton DNA.
SDS: SDS is a
of ion-type
. Its main functions are: (1) dissolve the lipids and proteins on the cell membrane, thus dissolving the membrane protein and destroying the cell membrane. (2) Dissolve the nucleoproteins in the cells. (3) SDS can be combined
protein
to become R-O-SO3-... A complex of R-proteins that denature and precipitate proteins. However, SDS inhibits the action of RNA, so it must be removed in the later extraction process to prevent interference in the next operation (when removing RNA with RNase).
3. 3mol/L NaAc (pH4.8) solution: NaAc's aqueous solution is alkaline and a large amount of ice acetic acid must be added in order to regulate pH to 4.8. So the solution is actually a buffer for NaAc-HAc. The NaAc solution with pH4.8 is intended to transfer the pumping solution of pH12.6 back to pH to neutral, so that the denatured granule DNA can be compounded and stable. The high-salt 3mol/L NaAc facilitates the condensation and precipitation of denatured large molecular chromosome DNA, RNA, and SDS-protein complexes. The former is because of the charge on the nucleic acid, reducing the rebuke force and polymerization with each other, the latter is because sodium salt and SDS-protein complex, can form a smaller sodium salt form complex, so that precipitation more complete.
4. Why precipitate DNA with waterless ethanol? Sedimentation of DNA with waterless ethanol is the most commonly used method of precipitation of DNA in experiments. The advantage of ethanol is that it can be mixed with water at any ratio, ethanol and nucleic acids do not have any chemical reactions, is safe for DNA, and is therefore an ideal sediment. DNA solutions are DNA that are stable in hydration, and when ethanol is added, ethanol robs the water molecules around the DNA, dehydrating the DNA and making it easy to polymerized. In general experiments, waterless ethanol, which is twice the volume, is mixed with DNA, and the final content of ethanol is about 67%. Instead of waterless ethanol (because waterless ethanol is much more expensive than 95 percent ethanol), it could also be replaced. However, the addition of 95% ethanol increases the total volume, and DNA dissolves to a certain extent in the solution, so DNA loss increases, especially when multiple ethanol deposits are used, which affects the yield. The compromise approach is to use 95% ethanol instead of a waterless b enzyme when the DNA is first precipitated, and the final precipitation step is to use waterless ethanol. IT can also selectively precipitate DNA with 0.6 times the volume of isopropyl alcohol. Usually at room temperature for 15-30 minutes.
5. Why add NaAc or NaCl to the final concentration of 0.1 to 0.25mol/L when precipitation DNA with ethanol? In a solution with a pH of about 8, the DNA molecule is negatively charged, plus a certain concentration of NaAc or NaCl, so that the negative charge on the Na-neutral DNA molecule reduces the estration of the same-sex charge between the DNA molecules, making it easy to The polymerization of each other to form DNA sodium salt precipitation, when the added salt solution concentration is too low, only part of the DNA to form DNA sodium salt and polymerization, resulting in incomplete DNA precipitation, when the added salt solution concentration is too high, its effect is not good. In precipitated DNA, due to the presence of too many salt impurities, affecting the DNA enzyme cutting and other reactions, must be washed or re-precipitated.6. Why use SDS and KAc to degrade ICNs after ICTs are added? The added RNase itself is a protein that must be removed in order to purify DNA, and SDS can make them SDS-protein complex precipitation, plus KAc to transform these complexes into SDS-protein complexes in the form of less soluble potassium salts, making precipitation more complete. It can also be used to saturate phenol, chloroform extract and re-precipitation, remove RNase. In the solution, someone can replace NaAc with KAc, which can also get better results.
7. Why is TE buffer generally used in the preservation or withdrawal of DNA? In
gene
, the main principle of selecting buffer is to consider the stability of DNA and the fact that the buffer composition does not interfere. Although the phosphate buffer system (pKa=7.2) and the boric acid system (pKa=9.24) are also in line with the physiological range of the intracellular environment (pH), which can be used as DNA preservation fluids, the type and number of phosphate ions will be precipitated with Ca2 plus Ca3 (PO4)2; Different types and quantity requirements, some require high ion concentration, some require low salt concentration, the use of Tris-HCl (pKa-8.0) buffer system, because the buffer is TrisH-/Tris, there is no interference of metal ions, so in the extraction or preservation of DNA, most of the Use Tris-HCl system, and TE buffer EDTA can more stable DNA activity.8. How do I choose the concentration of polyethyl glycol (6000) to precipitate DNA? Using PEG (6000) precipitation DNA, the concentration of PEG used by different sizes of DNA molecules is also different, peG concentration is low, selective precipitation DNA molecular weight is large, the concentration of PEG required by large molecules only about 1%, small molecules need PEG concentration of up to 20%. In this experiment, pBR322 proton DNA of 4.3kb was selectively precipitated, with 30% PEG of 0.4 ml per milliliter, with a final PEG concentration of 12%. PeG selective precipitation DNA has a resolution of approximately 100bp.9. How to use phenol and chloroform when removing DNA to remove proteins? Phenyl and chloroform are non-polar molecules, water is polar molecules, when the protein aqueous solution is mixed with phenol or chloroform, the water molecules between the protein molecules are squeezed by phenol or chloroform, so that the protein loses hydration and denaturation. After centrifugation, the density of the denatured protein is greater than that of water, so it is separated from the water phase and precipitated under the water phase, thus separating it from the DNA dissolved in the water phase. Phenols and chloroform
a
of solvents, retained in the lower layer. As surface denatured phenol and chloroform, in the role of removing proteins, each has advantages and disadvantages, phenol denaturation effect is large, but phenol and water phase has a certain degree of mutual dissolution, about 10% to 15% of the water dissolved in the phenolic phase, thus losing this part of the water phase DNA, and chloroform denaturation effect is not as good as phenol effect, but chloroform and water do not mix, will not take away DNA. Therefore, in the pumping process, the mixture of phenol and chloroform effect is best. Residual phenols are found in the water phase after the first withdrawal of phenols, which, because phenols and chloroform are mutually soluble, can be used as a second denatured protein of chloroform, at which point the phenols are taken away together. Phenols can also be mixed with chloroform (1:1) at the second withdrawal.
10. Why add a small amount of isoprene when extracting DNA with phenol and chloroform? In order to mix dna evenly, the container must be oscillat several times, when bubbles are easy to produce in the mixture, which prevents the full interaction between them. The addition of isosterol reduces molecular surface stress, thus reducing foam production during pumping. Chloroform and isoprene are generally used to have a ratio of 24:1. Phenols, chloroform and isotenol ratio of 25:24:1 (do not have to be pre-provisioned, can be added to a 24:1 chloroform and isosterol before use), while isotrol helps to phase, so that the centrifugal upper water phase, the middle denatured protein phase and the lower organic solvent phase to maintain stability.
11. Why saturate phenol with pH8 Tris aqueous solution? Can pink phenols be used? How do I keep phenols from being oxidized by air? Because phenol and water have a certain mutual solubility, phenol water saturation is designed to make it in the process of pumping DNA, do not absorb the sample contains DNA moisture, reduce DNA loss. Tris is adjusted to pH to 8 because DNA is stable under these conditions. Under neutral or alkaline conditions (pH5 to 7), RNA is more likely to free from the water phase than DNA, so DNA samples with less RNA content are available. Phenols stored in the refrigerator, easily oxidized by air and turned pink, are easy to degrade DNA and are generally not possible. To prevent phenol oxidation, dredging ethanol and 8-hydroxyquine can be added to the final concentration of 0.1%. 8-hydroxyquinone is a solid powder with a pale yellow color that not only antioxidant, but also inhibits the activity of DNase to a certain extent, it is a weak chelating agent of metal ions. Phenols saturated with Tris pH8.0 aqueous solution are best packed in small brown
reagents
bottles, covered with a Layer Tris aqueous solution or TE buffer, insulating air to fill the lid tightly, if possible, to fill nitrogen, to prevent oxidation from contact with air. Usually kept in a refrigerator at 4 degrees C or -20 degrees C, when used, open the lid after absorption and quickly cover, so that the phenolic invarmity, can be used for several months to precipitate the recovery of prosurfics when the phenol and chloroform pumping, only do ethanol precipitation OK?please see what they do! What is the role of using phenols to draw out cell DNA? The protein is denatured and the degradation of DNase is inhibited. When phenol is treated with homogeneous fluid, the protein molecule surface contains many polar groups similar to phenol because the protein and DNA bond have been broken. Protein molecules are dissolved in the phenolic phase, while DNA is dissolved in the water phase. Advantages of using phenols: 1. Effective denatured proteins; 2 inhibits the degradation of DNase. Cons: 1. Dissolves 10-15% of the water, thus dissolving a portion of poly(A) RNA. 2. The activity of RNase cannot be completely inhibited. What is the role of chloroform? Chloroform: Overcoming the disadvantages of phenols; Finally, with chloroform: remove trace phenols from the nucleic acid solution. When using phenol-chloroform to draw out cell genome DNA, it is common to add a little isopol to phenol-chloroform, why? Isosterol: Reduces bubbles produced during protein denaturation operations. Isosterol reduces surface stress, thereby reducing bubble production. In addition, isosterol helps to separate phases, so that the water phase of the upper layer containing DNA after centrifugation, the intermediate denatured protein phase and the lower organic solvent phase to maintain stability. Why add unit-priced cations when precipitation DNA with ethanol? When sequestration of DNA with ethanol, unit-priced cations, such as NaCl or NaAc, Na-neutral and negative charges on DNA molecules, are usually added to the solution, reducing the estrence of the same-sex charge between DNA molecules and making it easier to accumulate precipitation..