Extracting DNA from Living Things

February 19th, Tuesday 2019

10Rs did a procedure consisting of extracting DNA from a Kiwi Fruit.

Equipment:

For each group of students:

• Access to a water bath at 60℃ (optional)
• Test tube, 1, for each sample to be used
• Ice cold ethanol (IDA), 10 ㎤ for each sample to be used
• Wooden spill, straw, glass rod or including loop, 1 per sample

For the class- set up by a teacher

Blender for each material to be used or knives to chop material and a mortar and pestle material for each working group.

• Ice in a container to keep materials cool as necessary.
• Source/s or DNA (Kiwi Fruit)- to produce 10-20 ㎤ of blended material per sample.
• Table, salt, a pinch (or 1 ㎤) for every 300 ㎤ sample.
• Strainer for each material to be used.
• Detergent, 30 ㎤ for each 300 ㎤ of blended material to be processed.
• Protease, Eg: Pineapple juice, contact lenses cleaner, a pinch of meat tenderizer.

Procedure

SAFETY: Wear eye protection when handling the enzyme solution

Avoid skin contact with ethanol and with enzyme solutions or powders.

Wash any spills off your skin promptly.

Preparation

1. Chill your ethanol by placing in a container filled with ice for at least 2 hours, or overnight. Keep it on ice throughout the procedure.

Investigation

2. Make a thick ‘soup’ by blending your source material with a little table salt and some cold water. For example, use 100 ㎤ of split peas, with 200 ㎤  of cold water and a pinch of table salt (around 1 ㎤ ). Blend on high for 15 seconds.

3. Strain your ‘soup’ through a mesh strainer and collect the liquid part in a beaker.

4. Add 2 tablespoons (30 ㎤ ) of washing- p liquid and swirl to mix.

5. Let the mixture settle for 5-10 minutes. Some protocols recommend carrying out this stage in a water bath at 60 ℃. This may increase yield by increasing the breakdown of the cell and nuclear membranes or reduce yield it stimulates the action of DNase enzymes.

6. Pour the mixture into test tubes or other small glass containers, to make each about one-third full.

7. Add some protease enzymes to each test tube. You could use a pinch of meat tenderizer, a few drops of fresh pineapple juice, or some lens cleaning solution.

8. Tilt your test tube to 45° and slowly pour well-chilled ethanol (IDA) into the tube so that it forms a layer on top of your ‘soup juice’ - about the same volume as you have of ‘soup/juice’. Ethanol is less dense than water and will float on top. DNA is soluble in water, but salted DNA does not dissolve in ethanol and will form white clumps where the water and ethanol layers meet.

9. Use a wooden stick or a straw (or a glass rod) to collect the DNA. dip the stick into the tube and touch the white layer. Twirl the rod and the DNA should ‘spool’ onto the rod. DNA is a long, sti\ringy molecule. As you pull on one end of the strand, it pulls more DNA into the ethanol layer where it will precipitate.

10. Dry the sample on a paper towel if you want to measure the mass of product, or simply save the DNA by placing it in ethanol in any suitable small container with a lid.

Each cell in the human body contains 46 chromosomes. If you unraveled the DNA from each chromosome and put the 46 segments end-to-end, each cell would contain about 2 meters of DNA. each piece of DNA is around 4-5 cm long.

What happens at each step?

• Blending with salt and water: beaks the cells apart from one another and increases the surface area exposed to reagents such as detergent. It also begins to disrupt some of the cell walls of plant material.
• Adding salt: means the DNA is more likely to clump together when it meets the ethanol layer.
• Adding meat tenderizer: meat tenderizer commonly contains bromelain or papain-protease enzymes extracted from pineapple and papaya respectively. It will digest proteins associated with the DNA and so may help to purify the sample.

1. Using ice-cold ethanol and ice-cold water increases the yield of DNA. Low temperatures protect the DNA by slowing down the activity of enzymes that could break it apart. A cell's DNA is usually protected from such enzymes (DNases) by the nuclear membrane which is disrupted by adding detergent. DNases in the cytoplasm would destroy the DNA of viruses entering the cell. Cold ethanol helps the DNA to participate more quickly. Chill the ethanol in a screw cap plastic bottle in the prep room freezer. Below 4℃ ethanol is below its flashpoint so this is safe even if your freezer is not spark proof.

2. You can use a variety of substances for this extraction. The original of this protocol recommended split peas, but onions, and fish eggs or fish sperm (milt) are commonly recommended. It is important to check that your source material contains enough DNA. Kiwi fruit, strawberries, and bananas are often recommended, but it is usual pectin rather than DNA. Kiwi fruit temptingly contains protease unnecessary. Some foods (such as grapes) contain a lot of water and will make a watery 'soup' for good yields. The amount of DNA you will get will depend on the ratio of DNA to cell volume rather than the number of chromosomes in your material. Plant seeds (such as peas) contain a high proportion of DNA. (We used pieces of Kiwi fruit)

3. The salt added helps the DNA precipitate (as it clumps together) when it meets the ethanol phase.

4. It is important to allow time for each step to complete. The detergent must sit for at least 5 minutes to disrupt the cell membranes and nuclear membranes.

5. If you don't think you can see any DNA, dip your stick or rod into the surface of the 'soup' and then move it gently upward into the ethanol layer. Also, look closely at the ethanol layer for bubbles - sometimes clumps of DNA are loosely attached to the bubbles. If you can leave the mixture for 30-60 minutes, you may see more DNA precipitate.

6. Confirm that what you have is DNA by using a stain for DNA. (it may well be a mixture of DNA and RNA.) Confirm that what you have is not pectin by adding pectinase. If it dissolves it was pectin!