When the dilute salt solution of DNA is heated to 80~100 ℃, the double helix structure will be disintegrated, the two strands will be separated, and a random cluster will be formed. A series of physical and chemical properties will be changed, such as the increase of ultraviolet absorption value in 260 nm region, the decrease of viscosity, the decrease of buoyant density, and so on. The purpose of this experiment is to accurately understand the definition of the Tm value of DNA and the significance of determining the Tm value, and to master the method of determining the Tm value of DNA.
Operation method
ultraviolet spectrophotometry
Principle
When the dilute salt solution of DNA is heated to 80~100 ℃, the double helix structure will be disintegrated, the two strands will be separated, and a random cluster will be formed, and a series of physical and chemical properties will be changed. A series of physical and chemical properties are also changed: the ultraviolet absorption in the 260 nm region increases (color enhancement effect), the viscosity decreases, the buoyant density decreases, etc. The denaturation of DNA is characterized by an explosive process, and the denaturation occurs in a very narrow range. The temperature at which half of the double helix structure of DNA is lost is usually called the melting point or melting temperature (melting temperature) of the DNA, which is expressed as Tm. The Tm value of DNA is generally between 70 and 85 ℃. The size of the Tm value of DNA is related to the following factors: 1. the homogeneity of the DNA. The higher the homogeneity of the sample, the more the melting process occurs in a small temperature range. 2. G-C content. The higher the content, the higher the Tm, from the Tm value can be deduced from the GC content. The empirical formula is: ( G-C)% = ( Tm - 69.3 ) × 2.44. 3. Ionic strength of the medium. Generally, the melting degree of DNA is lower in the medium with lower ionic strength, and the range of melting degree is wider.The determination of Tm value and the study of denaturation conditions can provide us with information about the nucleotide composition of DNA, and also help to judge the purity and quantity of DNA.
Materials and Instruments
DNA Move 1 . Measuring Tm with a cyclic thermostat For more product details, please visit Aladdin Scientific website.
Sodium chloride Citric acid Ethylene glycol
UV Spectrophotometer Quartz Cup
Turn on the UV spectrophotometer and warm up for 20 min according to the operation procedure, set the circulating thermostat at 25 ℃, and perform the preliminary measurement of light absorption. Set the cycling thermostat at 25 ℃ for the preliminary measurement of light absorption. For homemade DNA, measure the A260 at 25 ℃ and dilute it to A260 with standard NaCl-citric acid solution. For homemade DNA, measure the A260 at 25 ℃ and dilute it with standard NaCl-citric acid solution to A260 = 0.4, and make sure that the final volume is suitable for the colorimetric cup (1 ml or 3 ml). and make sure the final volume is suitable for the colorimetric cup (1 ml or 3 ml).
Adjust the zero point of light absorption at 260 nm with standard NaCl-citric acid solution. The cuvette containing the diluted DNA solution to be measured is then placed in the sample chamber of the spectrophotometer and equilibrated for 3 minutes. Then place the cuvette containing the diluted DNA solution to be measured in the sample chamber of the spectrophotometer, equilibrate for 3 min and measure A260. Then the temperature was raised to 50 ℃, the cup was removed and the air bubbles were pushed out from the inner wall of the cup, and the A260 was measured. Measure A260. Continue to increase the temperature to 80 ℃, equilibrate for 5 min and measure A260. Increase the temperature by 2 ℃ each time, then equilibrate the temperature (5 min), and record the A260. A260. Continue in this manner until the A260 no longer increases.
2 . Measurement of Tm with a thermostatic bath
Set at least 6 thermostats (50 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃, 100 ℃), 95 ℃, 100 ℃). Preheat the UV spectrophotometer for 20 min according to the operation regulations. The concentration of DNA solution is controlled at A260 = 0.4. Take test tubes (at least 8 tubes), add 3 ml of DNA solution into each of them, put the cap on and incubate for 15 min. One test tube was placed at room temperature, two at 100 ℃, and one at the remaining temperature in a water bath for 15 min. One tube was placed at room temperature, two tubes were placed at 100 ℃, and one tube was placed in a water bath for the rest of the incubation period, and the tubes were cooled quickly after incubation. After incubation, the tubes were cooled rapidly, one at room temperature and the other at 100 ℃ for 10 min, while the other at 100 ℃ was slowly cooled to room temperature.
The A260 of the DNA solution was measured in each tube, and the slowly cooled tube was left at room temperature for at least 1 h before measurement. The slowly cooled tube was left at room temperature for at least 1 h before measurement.
3 . Results Processing
Calculate A260 ( t)/ A260 ( 25 ℃ ) at each temperature, and use this ratio to graph the temperature, connect the points to form a smooth curve. The midpoint of the increase in light absorption, Tm, was estimated by connecting the points to form a smooth curve. Calculation Calculate the G-C% content.