Extracting DNA from living things
Class practical or demonstration
You can extract DNA – to see what it is like – from some plant and some animal material using equipment and chemicals you might find in a kitchen. For more thorough analytical work, you need more control over the components of your chemicals, and it may be worth investing in a kit from one of the major suppliers. This is a rough and ready method that should give reasonable quantities of DNA from quite large quantities of material.
Lesson organisation
You can run this as a demonstration, or as small group work. Or you could prepare enough of each of several materials to allow groups to take samples from which they extract the DNA.
Apparatus and Chemicals
For each group of students:
Access to water bath at 60 °C (optional)
Test tube, 1, for each sample to be used
Ice cold ethanol (IDA), 10 cm3 for each sample to be used
Wooden spill, straw, glass rod or inoculating loop, 1 per sample
For the class – set up by technician/ teacher:
Blender for each material to be used
or knives to chop material and a mortar and pestle to grind material for each working group
Ice bath to keep materials cool as necessary (Note 1)
Source/s of DNA (Note 2) – to produce 10-20 cm3 of blended material per sample
Table salt, a pinch (or 1 cm3) for every 300 cm3 sample (Note 3)
Strainer for each material to be used
Detergent, 30 cm3 for each 300 cm3 of blended material to be processed
Protease, for example, pineapple juice, contact lens cleaner, pinch of meat tenderiser
Health & Safety
- Take care with ethanol IDA (see CLEAPSS Hazcard) – which is highly flammable and harmful through skin contact because of the presence of methanol.
- Protease (see CLEAPSS Hazcard) is harmful as a powder and irritant in solution. Wear eye protection and wash off skin promptly.
- Electrical equipment: Any electrical appliances used in the lab should be checked according to your employer’s systems. Use a blender dedicated for laboratory activities, not one that will be used later to process food for human consumption.
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 precipitate more quickly. Chill the ethanol in a screwcap plastic bottle in the prep room freezer. Below 4 °C 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 reported (see NCBE article Discovering DNA in the Links section) that the white strands produced here are usually pectin rather than DNA. Kiwi fruit temptingly contain protease that could help to digest the proteins surrounding DNA and make the addition of further protease unnecessary. Some foods (such as grapes) contain a lot of water and will make a watery ‘soup’. In this case, go back to the first step and add less water. You need an opaque cell ‘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.
3 The salt added helps the DNA to 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!
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
a Chill your ethanol by placing in a freezer for at least 2 hours, or overnight. Keep it on ice throughout the procedure (Note 1).
Investigation
b Make a thick ‘soup’ by blending your source material with a little table salt and some cold water (Notes 1, 2, 3). For example, use 100 cm3 of split peas, with 200 cm3 of cold water and a pinch of table salt (around 1 cm3 – Note 2). Blend on high for 15 seconds.
c Strain your ‘soup’ through a mesh strainer and collect the liquid part in a beaker.
d Add 2 tablespoons (30 cm3) of washing-up liquid and swirl to mix.
e Let the mixture settle for 5-10 minutes (Note 4). Some protocols recommend carrying out this stage in a water bath at 60 °C. This may increase yield by increasing the breakdown of cell and nuclear membranes, or reduce yield if it stimulates the action of DNase enzymes (Note 1).
f Pour the mixture into test tubes or other small glass containers, to make each about one third full.
g 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 contact lens cleaning solution.
h 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 (Note 5).
i 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, stringy molecule. As you pull on one end of the strand, it pulls more DNA into the ethanol layer where it will precipitate. (Note 3 and 5)
j 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.
Teaching notes
Each cell in the human body contains 46 chromosomes. If you unravelled the DNA from each chromosome and put the 46 segments end-to-end, each cell would contain about 2 metres of DNA. Each piece of DNA is around 4-5 cm long.
What happens at each step?
- blending with salt and water: breaks 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 tenderiser: meat tenderiser 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.
Investigations:
- which sources give the most DNA? how will you measure the sources and the product accurately for comparison?
- do you get better yields if you keep things cool throughout, or if you heat the blended mixture with detergent at 60 °C?
- which detergent works best?
- does the meat tenderiser make a difference? with every source?
- try extracting DNA from things which might not contain it
- how could you prove that this was indeed DNA? (what is the effect on it of stains which act on DNA such as toluidine blue or acetic orcein?)
Several companies produce kits for DNA extraction (for example Edvotek and NCBE – links below). The advantage of using kits is that any enzymes, salts, surfactants and buffer solutions provided will be pre-tested to ensure consistency from batch to batch, and hence reliability of the outcome of the procedure. They will also probably be cheaper than if you tried to source such materials directly. For a simple ‘bulk’ extraction like this, using many domestically available chemicals, it may not be necessary to use a kit. However, if you want clean DNA for further analysis, the kits are recommended. Some kits allow students to extract DNA from their own cheek cells and to encapsulate that DNA in a small plastic pendant (on a chain). Many students respond very positively to this personal dimension in the protocol, and it may make the procedure more memorable.
Health & Safety checked, March 2009
Web links
learn.genetics.utah.edu
This website provides a clear, simple procedure and useful FAQs.
www.edvotek.co.uk
Edvotek is a biotechnology education company providing kits and workshops to support teaching and learning in biotechnology. They produce a simple DNA extraction kit (What does DNA look like?) and a kit (Genes in a tube) for extracting DNA from student cheek cells and making the sample of DNA (in a microcentrifuge tube) into a pendant.
www.ncbe.reading.ac.uk
The NCBE has an international reputation for developing innovative educational resources and making sophisticated biotechnology techniques accessible to classroom teachers and students. They provide courses and materials for a wide range of biotechnology practicals – including a DNA pendant kit using which students can store the DNA collected from their own cheek cells in a glass vial. Their website also contains a link to a protocol for extracting DNA from frozen peas (at www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/unpublished.html).
The article Discovering DNA (in particular a paragraph headed DNA your onions?) on the NCBE site (www.ncbe.reading.ac.uk/DNA50/peadna.html) explains that fruits yield pectin rather than DNA.
(Websites accessed October 2011)