Experiment design
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.
Crude Extraction
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. On the other hand, 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. Therefore, the effect of different NaCl concentrations in the grinding solution on the crude DNA extraction needs further analysis.
Purification
Impurity removal methods include centrifugation, a 75°C water bath, and cold (4°C) 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.
Identification
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. Therefore, the diphenylamine method is not a precise identification method. Nucleic acids and proteins have peak absorption in the ultraviolet range at wavelengths of 260 nm and 280 nm, respectively. DNA concentration can be indirectly calculated through the absorbance at 260 nm: DNA concentration (μg/mL) = A260 × 100 × 50. Dodatno, 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.
Materials and Methods
Experimental Preparation
Oprema: 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, water bath, NanoDrop Lite ultramicroscopic nucleic acid/protein analyzer, fresh cauliflower.
Reagents: 95% ethanol frozen at -20°C, diphenylamine reagent.
Grinding Solution: Dissolve 1.01 g of Tris (hydroxymethyl) aminomethane in 5 mL of distilled water, adjust the pH to 8.0 using 2 mol/L hydrochloric acid, then add 0.876 (or 11.69) g NaCl, 3.72 g ethylenediaminetetraacetic acid (EDTA), i 2 g sodium dodecyl sulfate (SDS). After all the above chemicals are dissolved, make up the volume to 100 mL with distilled water.
Method Steps
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 min, na sobnoj temperaturi. “75°C water bath + 4°C settling” involves placing the test tube with the supernatant in a 75°C water bath for 10 min, followed by continued settling in a 4°C refrigerator for 10 min. “Only 4°C settling” means placing the supernatant in a 4°C refrigerator for 10 min.
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.
Recommendations
Optimization of Grinding Solution NaCl Concentration
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. Dodatno, higher NaCl concentrations can induce a salt effect, promoting protein precipitation, which is beneficial for DNA purification. Therefore, it is recommended to choose a grinding solution NaCl concentration of 2 mol/L.
Optimization of Impurity Removal Methods
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. Therefore, 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.
Optimization of Precipitate Treatment
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. Also, dilution of the solution may lead to a lighter color, making it difficult to observe distinct phenomena. Furthermore, diphenylamine may adsorb on each other, forming white small particles, hindering the color reaction. Međutim, 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.
