Common techniques of molecular cloning

1. Purification of nucleic acids

In all operations of molecular cloning, the most basic operation is the purification of nucleic acids. The key step is to remove protein, usually only phenol / chloroform. Chloroform can be used to extract the nucleic acid solution. This extraction can be performed whenever it is necessary to inactivate or remove some of the enzymes used in the clone for the next step. However, to purify nucleic acids from complex molecular mixtures such as cell lysates, most proteins must be digested with certain proteolytic enzymes before extraction with organic solvents. These broad-spectrum proteases include pronase and proteinase K, which are active on a variety of natural proteins. (1) Extraction with phenol and chloroform: the combined use of these two organic solvents is more effective than phenol extraction alone Better. Then chloroform extraction can remove trace phenol in nucleic acid products. ① Place the nucleic acid sample in a small centrifuge tube with a lid and add an equal volume of phenol / chloroform. ② Vortex to mix the contents of the tube to make it milky. ③ Centrifuge at 12000 × g for 15 seconds at room temperature. ④Move the water phase into another centrifuge tube, discard the two-phase interface and the organic phase. ⑤ Repeat steps ①-④ until no protein can be seen on the two-phase interface. ⑦According to the following nucleic acid concentration method, precipitate and recover nucleic acids.

2. Concentration of nucleic acids

The most widely used nucleic acid concentration method is ethanol precipitation. In the presence of a moderate concentration of monovalent cations, after adding a certain amount of ethanol, the formed nucleic acid precipitate can be recovered by centrifugation, and even DNA or RNA as low as pg can be recovered quantitatively. The recovered nucleic acid can be redissolved in the appropriate buffer at the required concentration.
For specific operation, add V / 10 monovalent cation salt storage solution 2V absolute ethanol to a small centrifuge tube containing sample, mix well, put in ice water bath for 15-30min, take out visual balance, 0-4 degrees, 12000g, centrifuge 10min. Aspirate the supernatant, and then add another 70% ethanol 0.5-1ml, 12000g, wash and centrifuge at 0-4 degrees for 2min. Aspirate the supernatant, drain the sediment with an oil pump or open the lid to dry, and dissolve in the appropriate volume of buffer.

Monovalent cation salt solution

*: When ammonium acetate is added, V / 2 of DNA solution needs to be added.

The selection of monovalent cation salts is mainly based on the following considerations: the use of ammonium acetate can reduce the co-precipitation of dNTPs, but in the future, the use of ammonium acetate should be avoided when phosphorylating nucleic acids, because the ammonium ion is T, and the polynucleotide kinase is strong Inhibitor. When precipitating RNA with a higher concentration of ethanol, LiCl is commonly used because LiCl is highly soluble in ethanol and does not co-precipitate with nucleic acids. For nucleic acid samples containing SDS, NaCl should be used, and the detergent must remain soluble in 70% ethanol. For the precipitation of DNA and RNA, sodium acetate (pH 5.2) is mostly used.

3. Quantification of DNA and RNA

The accurate method is ultraviolet spectrophotometry. However, this method requires that the nucleic acid sample is pure (that is, products without significant contaminants such as protein, phenol, agarose, or other nucleic acids, nucleotides, etc.). The light absorption at two wavelengths of 260 nm and 280 nm was measured with an ultraviolet spectrophotometer, and then, equivalent to 50 μg / ml double-stranded DNA according to IA260. 40μg / ml single-stranded DNA or RNA and 20μg / ml single-stranded oligonucleotide. Calculate your sample content. The ratio of the readings at 260nm and 280nm (A260 / A280) can reflect the purity of the nucleic acid. The A260 / A280 values ​​of pure DNA and RNA are 1.8 and 2.0 respectively. If the sample is contaminated with protein or phenol, the A260 / A280 will be significantly lower than this value, and the nucleic acid in the sample cannot be accurately quantified at this time. Samples can be purified before quantitative determination. People with extensive laboratory experience can only roughly determine the nucleic acid content in the sample based on the intensity of the ethidium bromide stained fluorescent band after the sample is electrophoresed, so they often do not quantify the nucleic acid by UV spectrophotometry.

4. Gel electrophoresis of nucleic acids and molecular weight reference

(1) Agarose gel electrophoresis can be used to separate, identify and purify DNA fragments. The method is simple and rapid, and can distinguish the mixture of DNA fragments that cannot be separated by other methods. The separated DNA can be stained with a low concentration of fluorescent dye (0.5μg / ml ethidium bromide), directly observed and detected under ultraviolet light as little as 1ng DNA.
The mobility of DNA through the agarose gel depends on the following parameters: â‘  The size of the DNA molecule: the rate at which the double-stranded DNA molecule passes through the gel is inversely proportional to the common logarithm of its molecular weight. According to this, a standard substance of known molecular weight and the DNA fragment of the molecular weight to be measured are electrophoresed at the same time, and the electrophoresis rate is compared to obtain the molecular size of the fragment to be measured. â‘¡Concentration of agarose: DNA fragments of a given size are expanded through agar with different concentrations at different speeds. Therefore, the use of different concentrations of gel can distinguish a wide range of DNA fragments of different sizes

Separation range of different concentrations of agarose gel

â‘¢DNA configuration: closed-loop (type I), open-loop (type II) and linear (type III) DNA of the same molecular weight pass through the gel at different rates. Generally, the mobility is type I> type III> type II. â‘£ Applied voltage: At low voltage, the mobility of linear DNA fragments is proportional to the applied voltage. However, when the pressure increases, the mobility of large-molecular-weight DNA fragments increases differently, so the effective separation range of agarose gel decreases with increasing voltage. In order to obtain the maximum resolution of DNA fragments, the voltage during gel electrophoresis should not exceed 5V / cm.

(2) Polyacrylamide gel electrophoresis can be used to analyze and prepare DNA fragments less than 1Kb in length

Effective separation range of DNA in polyacrylamide gel

Polyacrylamine gels are mostly used for vertical plate electrophoresis. When preparing the gel, first prepare 30% monomer mother liquor (29g acrylamide, 1g bisacrylamide, dissolve with water, and bring the volume to 100ml), then use it to prepare the required Concentration of gel. Add 30μl of tetramethylethylamine (TEMED) per 100ml of the above liquid, mix it, and then pour into the clean and leak-proof gel glass plate prepared in advance. When the gel is near the top, insert the appropriate " "Comb", let it polymerize at room temperature for 60 minutes. If the room temperature is too low in winter, you can put it in a 37-degree temperature oven to promote polymerization. After the polymerization is completed, pull out the "comb", fix the gel plate in the electrophoresis tank, pour 1 × TBE into the electrophoresis tank, rinse the sample hole and the bottom of the gel with a dropper to remove air bubbles, and you can add samples to electrophoresis. Generally, the voltage used is 1-8v / cm, and the migration of labeled rice is observed at any time. In polyacrylamide dissolved in 1 × TBE, the migration rate of the labeling dye is the same as the rate of the following DNA fragment

Migration of marker dyes in PAG *

* These numbers are the approximate size (in bp) of DNA fragments that co-migrate with the dye. At the end of electrophoresis, remove the glass plate from the cell and pry it apart carefully. The gel is immersed in ethidium bromide solution (0.5μg / ml1 × TBE) and stained. After 45 minutes, the electrophoresis result is observed under an ultraviolet lamp.

(3) Molecular weight reference substance In order to determine the size of the target DNA fragment, a molecular weight reference substance is often added next to the target DNA in the same gel. After electrophoresis and staining, the size of the target fragment can be quickly known under the UV lamp. The most commonly used molecular weight reference is Hind III digest, the size of each fragment is expressed in bp, respectively: 23130, 9416, 6557, 4361, 2322, 2027, 564, 125.

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