SUMMER RESEARCH PROGRAM FOR SECONDARY SCHOOL SCIENCE TEACHERS
The procedures involved in biotechnology implementation are predicated on the isolation of DNA from a tissue sample. [Content Standard E- Understandings about science/technology] 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.[Content Standard B- Structure and properties of matter]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.[Content Standard C- The cell] 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.
SDS (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.
EDTA (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.
NaCl (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.
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. TipsDNA 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.
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 15-20 min.
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 in freezer
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
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