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Purpose requirement
Learn the methods of crude extraction and identification of DNA, and observe the extracted DNA material.
Experimental principle
The solubility of DNA in sodium chloride solution changes as the concentration of sodium chloride changes. When the concentration of sodium chloride is 0.14mol / L, the solubility of DNA is the lowest. Using this principle, DNA dissolved in sodium chloride solution can be precipitated.
DNA is not soluble in alcohol solution, but some substances in cells can be dissolved in alcohol solution. Using this principle, DNA with fewer impurities can be further extracted.
DNA will be dyed blue when it meets diphenylamine (boiling water bath). Therefore, diphenylamine can be used as a reagent to identify DNA.
Experimental instruments and equipment
1. Iron stand 2. Glass rod (1 pc)
3. Filter paper 4. Measuring cylinder (100ml, 1 pc)
5. Beaker (1 for 100ml, 2 for 50ml and 2 for 500ml) 6. Test tube (2 for 20ml)
7. Funnel (1 pc) 8. Test tube clamp (2 pcs)
9. Gauze (15 pieces) 10. Centrifuge (1 set)
[Experimental Materials]
1. Chicken blood cell fluid (5ml ~ 10ml) 2. 95% ethanol (pre-cooled 24h)
3. Distilled water 4. Sodium citrate (mass concentration 0.1g / ml)
5. Sodium chloride (2mol / L and 0.015mol / L) 6. Diphenylamine
7. Perchloric acid 8. Acetaldehyde
9. Glacial acetic acid
Experimental procedure
Before the experiment, a chicken blood solution needs to be prepared. The method of preparation is to take 100 ml of a solution (anticoagulant) with a concentration of 0.1 g / ml of sodium citrate and place it in a 500 ml beaker. Inject the blood (about 180ml) from the slaughtered live chicken into the beaker while stirring with a glass rod to mix the blood with the sodium citrate solution to avoid blood clotting. Then, the blood was poured into the centrifuge tube, and centrifuged at 1000 rpm for 2 min, at this time, the blood cells settled on the bottom of the centrifuge tube. During the experiment, remove the clear liquid in the upper part of the centrifuge tube with a pipette to obtain chicken blood cell liquid (if there is no centrifuge, the blood in the beaker can be placed in the refrigerator and allowed to stand for one day to allow the blood cells to sediment).
1. Extract the nuclear material of chicken blood cells
Inject 5ml ~ 10ml of prepared chicken blood cell solution into a 50ml beaker. Add 20ml of distilled water to the beaker while stirring with a glass rod for 5min to accelerate the rupture of blood cells. Then, the blood cell liquid was filtered into a 500 ml beaker using a funnel with gauze, and the filtrate was taken.
2. Dissolve DNA in the nucleus
Add 40 ml of a solution with a concentration of 2 mol / L of sodium chloride substance to the filtrate, and shake the beaker to make it evenly mixed. At this time, the DNA carrier solution is in a dissolved state.
3. Precipitate viscous material containing DNA
Slowly add distilled water along the inner wall of the beaker, while stirring gently with a glass rod. At this time, there are filaments in the beaker. Pay attention to observe the color of the filaments. Continue to add distilled water, more and more viscous material will appear in the solution. When the viscous substance no longer increases, stop adding distilled water (the concentration of sodium chloride in the solution is equivalent to 0.14mol / L).
4. Filtration of viscous material containing DNA
Using a funnel with multiple layers of gauze, filter the solution from step 3 into a 500ml beaker. The viscous material containing DNA is left on the gauze.
5. Re-dissolve sticky DNA
Take a 50ml beaker and inject 20ml of a solution with a concentration of 2mol / L of sodium chloride into the beaker. Use blunt-end tweezers to clamp the viscous material on the gauze to the sodium chloride solution and stir continuously with a glass rod to dissolve the viscous material in the solution as much as possible.
6. Filter sodium chloride solution containing DNA
Take a 100ml beaker and filter the solution in step 5 with a funnel with two layers of gauze. Take the filtrate and dissolve the DNA in the filtrate.
7. Extract DNA with less impurities
To the above filtered solution, add 50ml of a cooled, alcohol volume fraction of 95% solution (using cooled alcohol, which has a better effect on DNA aggregation), and stir with a glass rod, there will be less impurities in the solution Filaments. Use a glass rod to roll up the filament and use filter paper to absorb the moisture. The main component of this filament is DNA. Pay attention to what color the filament is.
8. DNA identification
Take two 20ml test tubes, add 5ml of each solution of sodium chloride to a concentration of 0.015mol / L, put the filament into one of the test tubes, and stir with a glass rod to dissolve the filament. Then, add 4 ml of dianiline reagent to each of the two test tubes. After mixing, the test tube was placed in boiling water and heated for 5 min. After the test tube was cooled, observe and compare the color change of the solution in the two test tubes. Fill in the observation results in the "Experiment Report Book".
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