The experiment involves grinding and purifying plant materials to obtain a crude DNA extract. This extract is then used to identify the DNA present in the precipitate.
The solubility of crude DNA decreases initially and then increases with the increasing concentration of NaCl, reaching its minimum at a concentration of 0.14 mol/L.
Adding an ethanol solution to the crude DNA extract causes DNA to precipitate. At a NaCl concentration of 0.14 mol/L in the grinding solution, the solubility of DNA is minimal, which is not favorable for dissolving DNA during grinding but facilitates DNA precipitation. ในทางกลับกัน, at a NaCl concentration of 2 mol/L in the grinding solution, DNA solubility is higher, aiding in DNA dissolution, but the precipitation effect is not as good as in a low NaCl concentration grinding solution. ดังนั้น, the effect of different NaCl concentrations in the grinding solution on the crude DNA extraction needs further analysis.
Impurity removal methods include centrifugation, a 75°C water bath, and cold (4องศาเซลเซียส) settling. Centrifuging the crude DNA extract separates cell fragments and protein precipitates at the bottom of the centrifuge tube, while most of the DNA remains in the supernatant. By taking advantage of the difference in heat tolerance between DNA and proteins, maintaining the DNA extract at 75°C for 10 minutes in a water bath causes protein denaturation and precipitation. Cold settling at 4°C further precipitates proteins, polysaccharides, and other substances, achieving impurity removal.
Heating DNA in an acidic solution causes the degradation of 2-deoxyribose residues in the molecule, resulting in the formation of 2-deoxyribose and ω-hydroxy-γ-ketovaleraldehyde. The latter reacts with diphenylamine to produce a blue-colored compound. This reaction occurs without the need to dissolve DNA in a NaCl solution beforehand.
This experiment involves the crude extraction of DNA, and sugars, proteins, and derivatives in the DNA sample can also form various colored substances with diphenylamine. ดังนั้น, the diphenylamine method is not a precise identification method. Nucleic acids and proteins have peak absorption in the ultraviolet range at wavelengths of 260 นาโนเมตรและ 280 นาโนเมตร, ตามลำดับ. DNA concentration can be indirectly calculated through the absorbance at 260 นาโนเมตร: DNA concentration (มก./มล) = A260 × 100 × 50. นอกจากนี้, calculating the ratio of A260 to A280 can assess DNA purity. The A260/A280 ratio for pure DNA is 1.8, and this ratio significantly decreases in the presence of protein contamination.
By comparing the sediment quality, DNA concentration, and purity, as well as the color changes in diphenylamine identification between two sets of test tubes, the effectiveness of different experimental treatments for DNA extraction can be analyzed.
อุปกรณ์: Petri dishes, beakers, graduated cylinders, glass rods, cheesecloth, mortar and pestle, test tubes, test tube rack, funnel, electronic balance, analytical balance, centrifuge tubes, centrifuge machine, อ่างอาบน้ำ, NanoDrop Lite ultramicroscopic nucleic acid/protein analyzer, fresh cauliflower.
รีเอเจนต์: 95% ethanol frozen at -20°C, diphenylamine reagent.
Grinding Solution: ละลาย 1.01 g of Tris (hydroxymethyl) aminomethane in 5 มิลลิลิตรของน้ำกลั่น, ปรับ pH ไปที่ 8.0 using 2 โมล/ลิตร กรดไฮโดรคลอริก, จากนั้นเพิ่ม 0.876 (หรือ 11.69) g NaCl, 3.72 g ethylenediaminetetraacetic acid (อีดีทีเอ), และ 2 ก sodium dodecyl sulfate (เอกสารความปลอดภัย). After all the above chemicals are dissolved, make up the volume to 100 มล. ด้วยน้ำกลั่น.
① Grinding and Filtration: Grind cauliflower with grinding solutions of NaCl concentrations 0.14 mol/L and 2 mol/L, then filter to collect the supernatant.
② Impurity Removal: Treat the supernatant with three different methods for impurity removal, namely centrifugation, 75°C water bath + 4°C settling, or only 4°C settling. Centrifugation parameters are 3,000 r/min, 2 นาที, ที่อุณหภูมิห้อง. “75°C water bath + 4°C settling” involves placing the test tube with the supernatant in a 75°C water bath for 10 นาที, followed by continued settling in a 4°C refrigerator for 10 นาที. “Only 4°C settling” means placing the supernatant in a 4°C refrigerator for 10 นาที.
③ Precipitation: Pour an equal volume of cold ethanol into the test tube, and let it settle. Once white flocculent material appears, use tweezers to remove the precipitate, air-dry, and weigh.
④ Identification: Determine the DNA concentration and analyze the DNA purity of the samples using the NanoDrop Lite ultramicroscopic nucleic acid/protein analyzer. Qualitative identification is done using a diphenylamine reagent. The samples are not dissolved before diphenylamine identification or dissolved in NaCl solution with a concentration of 2 mol/L of different volumes.
The experimental results show that, whether in terms of DNA concentration or purity, the DNA extraction efficiency is better with a grinding solution NaCl concentration of 2 mol/L compared to 0.14 mol/L. This suggests that when the NaCl concentration is 2 mol/L, the solubility of DNA is higher. Although a NaCl concentration of 0.14 mol/L is more favorable for later DNA precipitation (compared to 2 mol/L), this effect is weak due to the action of ethanol. นอกจากนี้, higher NaCl concentrations can induce a salt effect, promoting protein precipitation, which is beneficial for การทำให้ดีเอ็นเอบริสุทธิ์. ดังนั้น, it is recommended to choose a grinding solution NaCl concentration of 2 mol/L.
The experimental results indicate that different impurity removal methods have their pros and cons, and some DNA loss occurs while removing proteins. Analyzing from DNA purity, centrifugation is optimal; from DNA concentration, 4°C settling is optimal; considering cost and operational difficulty, 4°C settling is optimal. ดังนั้น, the impact of the three impurity removal methods on the experimental results is similar, but 4°C settling is cost-effective, convenient to operate, and practical when time and cost are limited.
The experimental results show that the identification results are better when “not dissolving precipitates.” This may be because diphenylamine forms a milky solution when in contact with water, hindering the observation of the gray-blue color. อีกด้วย, dilution of the solution may lead to a lighter color, making it difficult to observe distinct phenomena. นอกจากนี้, diphenylamine may adsorb on each other, forming white small particles, hindering the color reaction. อย่างไรก็ตาม, it is suggested to directly add the precipitate to the diphenylamine reagent for identification or dissolve the precipitate in a small amount of 2 mol/L NaCl solution for identification (the recommended volume is one-fourth of the diphenylamine volume). This approach avoids a decrease in identification sensitivity caused by dissolving the precipitate.
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