Extraction of DNA from White Onion
The procedures involved in biotechnology implementation are predicated on the isolation of DNA from a tissue sample. This laboratory exercise is designed to give you the opportunity to extract DNA from onion tissue. The technique used is quick and easy for both you and your students. The DNA that is isolated can be digested using various endonucleases, followed with an electrophoresis of the digest. We use an onion because of its' cost, abundance and low starch content. You will make the onion filtrate from onion treated with salt, distilled water, and dishwashing DNA to be more clearly seen. The SDS detergent causes the cell membrane to break down by emulsifying the lipids and proteins of the cell and disrupting the polar interactions that hold the cell membrane together. The detergent then forms complexes with these lipids and proteins, causing them to precipitate out of solution. The use of NaCl salt shields the negative phosphate ends of the DNA which allows these ends to come closer so they can precipitate out of a cold 95% ethyl alcohol solution. You will be altering the filtrate so that you can "spool" DNA out when it precipitates. The DNA is soluble in the detergent solution but is insoluble in the alcohol. When you add the chilled alcohol, the DNA will come out of solution and easily spool on a glass rod. At the end of the exercise you will find a flow chart summarizing the steps and solutions required.
The procedure has three basic steps:
Homogenization which involves heating and blending the onion tissue in order to break down the cells. The heat treatment softens the phospholipid in the cell membrane and denatures the DNAse enzymes which if present, would cut the DNA into small fragments so that it would not spool. The onion tissue is mixed in a blender with homogenization media, which breaks down the cell wall, cell membrane and nuclear membrane allowing the release of DNA.
Deproteinization which involves adding a protease enzyme Papain - a common enzyme used to clean soft contact lenses. This will denature the proteins clinging to the DNA making the molecule flexible and easy to spool. Precipitation of DNA which involves adding ethanol alcohol which causes every component in the filtrate to stay in solution except DNA. The DNA will gather at the interface of the filtrate and ethanol and can be spooled out with a glass rod.
(Sodium Dodecyl Sulfate) is a biological detergent which causes the cell membrane to break down further and emulsifies the lipids and proteins of the cell by disrupting the polar interactions that hold the cell membrane together. The detergent forms complexes with these lipids and proteins causing them to precipitate out of the solution. SDS is the major ingredient in laundry detergent.
(Ethylenediamine tetracetic acid) weakens the cell by binding the divalent cations (Mg++ and Ca++) which are needed for membrane stability. This further aids in breaking open the cells of the onion.
(Sodium chloride) enables nucleic acids to precipitate out of an alcohol solution because it shields the negative phosphate end of DNA causing the strands to come closer together and coalesce.
- DNA is found in the nucleus of membrane-bound cells. The membranes are lipid and protein in composition. The cell membranes must be lysed in order to release the DNA.
- DNA is a polymer made up of repeating chains of nucleotides. The sugar and phosphate components of DNA (the backbone) are both readily soluble in water.
- The phosphate groups on the outside of DNA carry a negative charge. These negative charges are attracted to and are neutralized by cations such as sodium. When sodium is added to DNA it forms a protective "shell" around it. On the other hand, protein molecules precipitate from solution in the presence of salt.
- DNA is insoluble in ethanol (ethyl alcohol). As ethanol is added to a solution containing DNA, the DNA will come out of solution and stick to whatever is around.
- At room temperature DNA begins to denature by the action of DNase (present in cell extracts). DNA extraction procedures must be carried on in ice.
To use DNA for cloning or restriction digests, wash with 95% ethanol, then 70% ethanol. Air dry and resuspend in 500ul TE buffer. Heat in a 60 C water bath for 10 minutes to denature potential DNases. Store at -20 C.
DNA clings to glass - negative charge of DNA is attracted to positive charge in the silica of glass. Therefore, use plastic tubes for the spooling part of this lab
You can make simple glass rods by heating the ends of glass pasteur pipettes and pushing the end to make a small hook. These make dandy rods to spool and hook up the DNA.
A similar procedure to this one can be used to extract DNA from animal tissue such as calf thymus. Thymus glands from calves are sold in butcher shops and gourmet grocery stores as "sweetbreads". You can use either fresh or frozen.
- Place 100 ml of homogenizing solution in a beaker heat the solution in a water bath until it reaches 60 C
- Mince the onion and add to the solution when it has reached 60 C. Stir and let sit for 15 minutes. Try not to let the temperature go much above 60 C. The temperature is intended to denature proteins that would break up the DNA into small segments.
- After the heat treatment, immediately place the beaker into an ice bath for 5 minutes. Swirl the solution gently to allow even cooling throughout. This step slows down the break down of DNA.
- Pour the contents of the beaker into the homogenizer and blend as per the flow chart.
- Filter the homogenate through cheesecloth draped over a clean beaker.
- Pour some of the filtrate into a large test tube. Hold your test tube with filtered homogenate at an angle, gradually pour twice the volume of ice cold alcohol down the wall of the test tube as there is homogenate present.
- Watch what happens. You should see some stringy substance precipitate out. This is DNA. When it looks very stringy, place a glass rod into the tube so that the end of the rod is just bellow the upper layer of liquid( alcohol ) and try to spool the DNA. It should look very clear and glistens around the glass rod. Using a glass stirring rod, gently but quickly twirl the rod into and out of the 2 layers. Gently lift the rod out of the tube and observe any substance attached to the rod.
- This DNA represents all the DNA found in the onion cells. The chromosomes were broken in the process and the DNA precipitated due to the chemical treatment.
Extraction of DNA from Onion
Dice an onion into small pieces
weigh out 50g of onion
place onion into a 250 ml beaker
add 100 ml of homogen. medium
incubate in a 60 C water bath
for 15 min.
chill quickly in an ice bath (15-20 C)
handle the DNA gently, not rough
pour chilled preparation into blender
homog. for 45 sec. at low speed
homog. for 30 sec. at high speed
pour into a 1000 ml beaker
allow to stand in ice bath for
pour through 4 layers of cheesecloth
over a 500 ml beaker in ice
slowly add cold 95% ethanol down the
side of the beaker 80 ml
spool out DNA in one direction only
dry the DNA with paper towel
and suspend in TE buffer, store
5% SDS (50g/L) --------------------------------- 0.15M NaCl (8.8g/L)
0.15M sodium citrate (43.7g/L) ------------------ 0.001M EDTA (0.5M stock, 2ml/L)
TE Buffer pH 8.0
0.01M Tris-HCl pH 8.0 --------------------------- 5 mls of 2M stock
0.001M EDTA pH 8.0 ---------------------------- 2 mls of 0.5M stock