How do scientists extract DNA? Step by Step! DNA extraction
Hello in this article I will be telling you exactly how to extract DNA from a plant cell down to the exact chemicals to use. I have done this procedure in the laboratory before and it is pretty straight forward. I thought I’d make this article for other students and for anyone who is interested in how scientists actually extract intact strands of DNA. I will try to explain each step to make it easier for anyone understand.
- DNA extraction buffer: 0.1M NaCl , 0.01M EDTA (pH 8.0), 0.3M Tris (pH 8.0)
- 5M NaCl
- Absolute ethanol
- Rnase A (10mg/ml)
Explaining The Solutions used!
DNA extraction buffer is used to maintain the pH of a solution and to stop DNases from working. DNase is an enzyme that breaks down a DNA molecule therefore it is vital that we prevent DNases from breaking down the DNA that we wish to extract. Tris and EDTA (Ethylenediaminetetraacetic acid) maintain the pH of the solution at 8.0 as the optiumum pH for DNAases is 7.0 thus, maintaining the pH at 8.0 prevents DNases from working. EDTA is a chelating agent meaning, EDTA will bind to metal ions in a solution, such as the metal magnesium. The metal ion magnesium, is a co-factor of the enzyme DNase and is essential for its function. EDTA binds to magnesium and prevents DNases from using magnesium as its co-factor; DNases are further prevented from catalysing any reactions.
Phenol and Chloroform are strong protein denaturing solvents. DNA is wound up in a cell by proteins called histones. In order to extract the DNA from the cell histones must be removed from the DNA molecule. Removing the histones allows the DNA strand to uncoil and be free in the cell. Chloroform and phenol will bind to the proteins removing them from the DNA strands.
Absolute ethanol is used to precipitate the DNA out of the final solution. When ethanol is added to a solution containing DNA it will show up as a white strand and it visible to the naked eye.
An RNase is an enzyme that breaks down an RNA molecule in the same way that a DNase will break down a DNA molecule. RNases are added to the final solution to ensure that no RNA is in the solution as it may interfere with the DNA in the solution when trying to extract.
(see figure 1 for help with understanding centrifugation and the different layers produced)
- Place 5ml of 10% SDS + 20ml DNA extraction buffer into a blue sterile plastic tube (50ml) in a rack. SDS is used to destroy cell membranes thus opening up cells.
- Chill mortar and pestle with small amount of liquid nitrogen. Then put pre-frozen leaf (I used spinach leaves from my local supermarket!) sample into pre-chilled mortar.
Liquid nitrogen is used to prevent any metabolic processes in a cell from working. A pre-frozen leaf and a pre-chilled mortar is used because everything used in this experiment must be of similar temperature. If a piece of equipment is used to touch the sample it must be of similar temperature at this stage, a difference in temperature will cause thawing and in the end destroy the DNA sample.
- Grind leaf sample in liquid nitrogen to a fine powder using the mortar and pestle. The leaf must be ground into a very fine powder in order for the extraction to work! The cells will be broken up and the DNA will be allowed to diffuse out of the cell as the cell membrane is destroyed.
- Cool metal spatula in liquid nitrogen and then spoon leaf powder into tube containing DNA extraction buffer prepared in step 1. The cooling of the spatula is to prevent thawing of the sample!
- Mix well by slow inversion of the tube, then top up tube with chloroform and mix well. Leave sample to stand for 10 minutes with occasional mixing.
- Centrifuge sample at 3,000 rpm at room temperature for 10 minutes.
Centrifugation is a process where you spin a sample at a specific rate and time to separate different layers within a solution according to there molecular weight.
- Use the wide end of an inverted sterile (10ml) pipette to remove the aqueous layer from tube containing sample to a fresh (50ml) plastic tube.
The aqueous layer is the layer containing the DNA, the rest of the solution is waste. This waste includes cell organelles, chloroform and proteins (histones).
A wide ended inverted pipette is used to avoid shear stress on the DNA strand. A normal ended plasic pipette tip would cause to much stress on the DNA molecule because the end of the pipette tip is very narrow; destroying the DNA molecule.
- Add 10ml phenol. Mix. Leave at room temperature for 5 minutes, mixing occasionally.
- Top up with chloroform. Mix. Leave at room temperature for 5 minutes, mixing occasionally.
- Centrifuge at 3,000 rpm for 10 minutes. Remove aqueous phase into fresh plastic tube
Dispose of phenol into waste jar.
- Add chloroform to fill tube. Mix and leave to stand at room temperature for 5 minutes.
- Centrifuge at 3,000 rpm for 10 minutes. Remove supernatant and place in a fresh plastic tube with inverted sterile 10ml wide ended pipette.
- Top up supernatant with absolute ethanol, mix gently by inversion and watch for appearance of strands of DNA (whitish in color).
- Centrifuge genomic DNA at 2000 rpm for 5 minutes. Tip supernatant down sink and repeat spin twice. Pipette off final supernatant.
- Resuspend pellet in 5ml of THE pH 8.0 + 25ml Rnase A (Dnase free) and incubate at 40C
There you go! How to extract DNA! As I said in the introduction I have done this procedure before and successfully extracted DNA. Any questions just leave a comment.
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