(1) Identification of nucleic acids
1. Concentration Identification The quantitative identification of nucleic acid concentration can be carried out by ultraviolet spectrophotometry and fluorescence spectrometry.
(1) Ultraviolet spectrophotometry:
Ultraviolet spectrophotometry is based on the fact that the bases in the nucleic acid molecule have a certain ultraviolet absorption characteristic, and the maximum absorption wavelength is between 250 nm and 270 nm. When these bases form nucleotides with pentose and phosphoric acid, their maximum absorption wavelengths are unchanged. After the nucleic acid is composed of nucleotides, its maximum absorption wavelength is 260 nm, which lays a foundation for determining the concentration of nucleic acid in the solution. Under ultraviolet light having a wavelength of 260 nm, the optical density of one OD value corresponds approximately to 50 μg/ml of double-stranded DNA, 38 μg/ml of single-stranded DNA or single-stranded RNA, and 33 μg/ml of single-stranded oligonucleotide. If the concentration of a single-stranded oligonucleotide molecule of a known sequence is to be accurately quantified, it must be calculated in accordance with Lambert-Beer's law in combination with its actual molecular weight and molar absorptivity. If the DNA sample contains salt, the A260 reading will be too high. A310 should be determined to subtract the background, and the difference between A260 and A310 should be used as the basis for quantitative calculation. Ultraviolet spectrophotometry is only used to determine nucleic acid solutions at concentrations greater than 0.25 [mu]g/ml. Shanghai Chuangsai Technology provides 163795-75-3, SYBR Green 1, fluorescent dye, SYBR Green I nucleic acid gel stain; BR, product number: D23-RS1151-100 microliters, price 416 yuan.
(2) Fluorescence method:
Fluorescence spectrophotometry uses the nucleic acid fluorescent dye ethidium bromide (EB) to embed the base plane, so that the non-fluorescent nucleic acid emits orange-red fluorescence under ultraviolet excitation, and the fluorescence intensity integral is proportional to the nucleic acid content. The sensitivity of the method can reach 1 ng to 5 ng, which is suitable for quantitative analysis of low concentration nucleic acid solution. In addition, SYBR Gold is a new ultra-sensitive fluorescent dye that detects less than 20 pg of double-stranded DNA from an agarose gel. Shanghai Chuangsai Technology provides ethidium bromide (EB), Ethidium bromide (EB), 0.98, 1239-45-8, product number: C84-4222-1G, and the price is 300 yuan.
2. Purity identification Ultraviolet spectrophotometry or fluorescence spectrometry can be used for the purity identification of nucleic acids.
(1) Ultraviolet spectrophotometry:
Ultraviolet spectrophotometry mainly determines the presence or absence of protein contamination by the ratio of A260 to A280. In TE buffer, the pure DNA had an A260/A280 ratio of 1.8 and the pure RNA had an A260/A280 ratio of 2.0. Both the increase and decrease in the ratio indicate impure. Both the protein and the phenol added in the nucleic acid extraction reduced the ratio. The high absorption peak of the protein at 280 nm and the high absorption peak of phenol at 270 nm can identify whether it is mainly protein contamination or phenol contamination. RNA contamination can cause the ratio of DNA products to be higher than 1.8, so a DNA solution with a ratio of 1.8 is not necessarily a pure DNA solution, and may be contaminated with protein, phenol and RNA, and needs to be identified by other methods. The ratio of A260/A280 is a good indicator of the degree of protein contamination, and 2.0 is a hallmark of high quality RNA. However, it should be noted that due to the different secondary structure of RNA, the reading may have some fluctuations, generally between 1.8 and 2.1 is acceptable. In addition, the pH of the solution used to identify RNA purity affects the A260/A280 reading. For example, the A260/A280 ratio of RNA in aqueous solution is 0.2 to 0.3 lower than its reading in Tris buffer (pH 7.5).
(2) Fluorescence method:
The result of nucleic acid electrophoresis traced with a fluorescent dye such as ethidium bromide can be used to determine the purity of the nucleic acid. Because DNA molecules are much larger than RNA, electrophoretic mobility is low; RNA has the most rRNA, accounting for 80%-85%, tRNA and nuclear small RNA accounting for 15%-20%, and mRNA accounting for 1%-5%. Therefore, the total RNA can exhibit three characteristic bands after electrophoresis. In the prokaryote, there are clearly visible 23S, 16S rRNA bands and a relatively diffuse fast-migrating band composed of 5S rRNA and tRNA. In eukaryotes are 28S, 18S rRNA and a band consisting of 5S, 5.8S rRNA and tRNA (Figure 6-1). The lack of mRNA and the size of the molecules are generally invisible. By analyzing the results of nucleic acid gel electrophoresis using ethidium bromide as a tracer dye, we can identify the presence or absence of RNA interference in DNA products, and also identify the presence or absence of DNA contamination in RNA products.
3. Integrity Identification Gel electrophoresis is routinely used.
(1) Gel electrophoresis:
The results of nucleic acid gel electrophoresis using ethidium bromide as a tracer dye can be used to determine the integrity of nucleic acids. Genomic DNA has a large molecular weight and is very slow to move in an electric field. If a small molecule DNA fragment is degraded, it can be significantly expressed on the electropherogram. The complete total RNA electropherogram without degradation or degradation, except for the characteristic three bands, the fluorescence intensity integral of the three bands should be a specific ratio. The nucleic acid band with large sedimentation coefficient has large molecular weight, low electrophoretic mobility and high fluorescence intensity integral; on the contrary, the molecular weight is small, the electrophoretic mobility is high, and the fluorescence intensity integral is low. Generally 28S (or 23S) RNA has a fluorescence intensity about twice that of 18S (or 16S) RNA, which otherwise suggests RNA degradation. If there is a colored band near the loading slot, it indicates DNA contamination.
(2) Other methods:
If necessary, RNA can also be analyzed by special tests, such as small-scale first-strand cDNA synthesis reactions, radio-labeled oligo-deoxythymidine oligo (dT) probes for Northern hybridization, and Northern hybridization of known size mRNA. In addition, with the rapid development of capillary electrophoresis and biochip technology, the means of separation, purification, identification and recovery of nucleic acids are increasingly abundant.
(2) Preservation of nucleic acids
The structure and properties of nucleic acids are relatively stable, and it is not necessary to prepare fresh nucleic acid samples every time, and the nucleic acid samples prepared at one time can often meet the needs of multiple experimental studies, so it is necessary to investigate the storage environment and conditions of nucleic acids. As with isolation and purification, the storage conditions of DNA and RNA are also different depending on the nature.
1. Preservation of DNA
For DNA, it can be stored in TE buffer at -70 ° C for several years. The pH value of TE is 8, which can reduce the deamination reaction of DNA, and the DNA is easily denatured when the pH is lower than 7.0. EDTA acts as a chelating agent for divalent metal ions to inhibit DNase by chelation of divalent metal ions such as Mg2+ and Ca2+. The activity of low temperature is beneficial to reduce various reactions of DNA molecules; double-stranded DNA is very inert due to structural characteristics, and can be stored for a long time at 4 ° C; adding a small amount of chloroform to DNA samples can Effectively avoid contamination of bacteria and nucleic acids. Shanghai Chuangsai Technology provides diethyl pyrocarbonate, DEPC (Diethylpyrocarbonate), 1609-47-8, commodity number: B11000132-100ml, and the price is 680 yuan.
2. RNA preservation
The RNA can be dissolved in 0.3 mol/L sodium acetate solution or double-digested water, and stored at -70 ° C to -80 ° C. If RNA is dissolved in diethyl pyrocarbonate (DEPC) or RNasein or vanadyl-ribonucleoside complex (VRC) is added to the RNA solution, Inhibition of RNase degradation of RNA and prolonged storage time. In addition, the RNA precipitate is dissolved in a 70% ethanol solution or a deionized formamide solution and can be stored at -20 ° C for a long period of time. Among them, the formamide solution can avoid the degradation of RNA by RNase, and the RNA is very soluble in the formamide solution, and its concentration can be as high as 4 mg/ml. It should be noted that the addition of these so-called RNase inhibitors or organic solvents is only a temporary need to be preserved, and if they have an impact on subsequent experimental research and application, they must be removed. Shanghai Chuangsai Technology provides RNase inhibitor, RNasin Inhibitor, commodity number: B11000504-10000u, price 826 yuan.
Since the mechanical shearing force generated by repeated freezing and thawing has a destructive effect on DNA and RNA nucleic acid samples, in practice, a small amount of nucleic acid is very necessary.
(C) the application of nucleic acids
The isolated and purified nucleic acid sample can be used for the study of the function and properties of the nucleic acid itself, and can also be widely applied by its functions and properties, such as genetic engineering and protein engineering using nucleic acid molecules as the original material. It can be said that most molecular biology techniques, including polymerase chain reaction, molecular hybridization of nucleic acids, DNA recombination and expression technology, are based on nucleic acid molecules, using the base pairing principle of nucleic acid molecules and nucleic acids. Performing one or more enzyme modifications. The vast majority of molecular biology techniques are essentially the analysis of DNA and RNA. Without the isolation and purification of nucleic acids, molecular biology techniques have no basis for research and application.
Shanghai Chuangsai Technology has excellent performance, interleukin cytokines, fetal bovine serum, electrophoresis equipment scientific instruments, raw material drug standards, chemical reagents, cell culture consumables, Shanghai Chuangsai, mass products special promotions, welcome to inquire!
Iron Clip Pen ,Ball Pen ,Ballpoint Pen,Waterproof Gel Pen
Tonglu shangmei pen industry co.,ltd , https://www.shangmeipenindustry.com