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|  | Excerpts from AP Biology Teachers Discussion Group:
Tip: "One of the tricks I've discovered is to use wood splints for spooling, rather than glass rods. We save it in capped vials in 50 percent ethanol for years. It stays in the refrigerator (although that probably is not necessary) and keeps looking cool. I think Doris Helms' lab manual says to use 60° C rather than 40 to 50."
-- Barbara Beitch, Hamden Hall Country Day School, Hamden, Connecticut. 1/29/99
Tip: "Try doing your onion DNA isolation without chopping them in a blender -- if you did that. I use just a sharp knife and it works pretty well. Also you might try kiwi fruits. Smash them with a large spoon in the detergent solutions you use for onions and they work really well."
-- Doug Herman, East High School, Sioux City, Iowa. 1/29/99
Question: " Is there an 'over-the-counter' substitute for sodium dodecylsulfate (SDS) used in DNA extraction protocols?"
Answer: "I use regular old liquid dish soap. It does the trick."
-- Bob Heun, Brooks School, North Andover, Massachusetts. 12/2/99
Question: "Can you help on DNA extraction protocol?"
Answer 1: "I use dog testes (for DNA extraction). I usually get a big batch and freeze them and they have lasted for years. Be sure to ask for non-preserved ones from your vet and from mature dogs (not puppies). I bought the EDTA and SDS the first year and still have it. It takes so little that I have a lifetime supply and, in fact, I keep the solutions refrigerated and often use them the next year with great results."
-- Charlotte Freeman, Girls Preparatory School, Chattanooga, Tennessee. 11/3/99
Answer 2: " - Place a small piece calf thymus (sweetbreads from the store) into a mortar. Cut the sample with a pair of scissors into smaller pieces. Grocery stores carrying pigs feet, calves brains, and such usually have sweetbreads; in Texas, Fiesta Foods and HEB carry them.
- Add 10 mL of 0.9 percent NaCl solution and grind with the pestle for 2-5 minutes.
- Strain the solution through 3 to 4 layers of cheesecloth into a test tube. (0.0 g of NaCl in 100 mL of water). Keep the tube with the suspension.
- Add 1.5 mL of 10 percent sodium dodecylsulfate (SDS) to cell suspension and mix. Lacking SDS add 3 or 4 drops of Dawn dishwashing liquid (it contains SDS). The SDS lyses the cell membrane by dissolving the lipoproteins in the cell membrane.
- Measure the total sample volume, then measure out two times that amount of ice cold 95 percent ethanol. (I keep it in the freezer prior to using.) Down the side of the tube, gently add the ethanol to the suspension.
- Use a glass stirring rod to gently stir the mixture until the DNA begins to precipitate at the interface. It usually precipitates as you add the EtOH. Then twist the glass rod to spool the DNA onto the rod. The precipitated DNA can be transferred to a new tube containing 95 percent ethanol and stored indefinitely in the freezer. From Texas Biotechnology Teacher Enhancement Project -- Texas A & M."
-- Nancy Hein, Hawley High School, Hawley, Texas. 11/3/99
Answer 3: "If you are determined to try DNA extraction with thymus gland, try an ethnic market. In the Houston area, I buy them at Fiesta. The other day I was amazed to see it for sale at Kroger. (Under its pseudonym of 'sweetbreads,' of course.) I did a DNA extraction with thymus in my classes for several years, then switched to doing it with onions. It works better because you can always get fresh material. Thymus that has been around too long or has been frozen and thawed several times doesn't work well. Bacterial cultures can also be used as a source of DNA."
-- Alexa Noble, Oak Ridge High School, Conroe, Texas. 11/3/99
Answer 4: "I got a DNA isolation procedure from Scientific American: it works very well for spooling DNA (Scientific American Set 1998, The Amateur Scientist 1998). You could probably clean the DNA up with a salt and ethanol precipitation to run it on a gel. I am embarrassed to admit that I just realized this may not be possible in a high school laboratory because you need a medium speed (10,000xg) centrifuge. Scientific American had directions on how to make one from a blender, I don't know if a clinical centrifuge would work. I don't know how well this will work with spooled DNA -- I will be trying it -- but in research labs people precipitate DNA a lot to change the suspension solution or just to remove impurities. So, if you are still interested, to precipitate DNA: - Take your spooled gob, air dry, and then dissolve it in water or saline. If you add 1mM EDTA and use a buffered solution that may stabilize the DNA, but it is probably not necessary. The DNA strands are long, so if you mix it vigorously the strands will break. I do not think this will be a problem, but it may cause a smear on the gel, so be gentle.
- Add sodium acetate (pH5.0) to get a final sodium acetate concentration of 0.3M -- most people use a 3M sodium acetate stock solution, buffered with acetic acid to pH5.0. Then if you have 1 mL of DNA, add 0.1ml of the concentrated acetate.
- Then, add ethanol to a final concentration of near 70 percent (2.5 mL for 1 mL of DNA). You will start to see the DNA come out of the solution and you can chill it in the freezer to improve the precipitation.
- Finally, centrifuge it at 10,000xg for five minutes or so -- maybe longer in a clinical centrifuge would work -- and you have a glossy pellet of clean DNA that you can re-suspend in a buffer for electrophoresis or enzyme digestion.
Total genomic DNA will often give you a smear because the DNA molecules are so long and broken randomly."
-- Joan Kiely, SUNY, Stony Brook, New York. 2/9/00 and 2/11/00
Answer 5: "I have this recipe thanks to Dr. Jeff Smith of the Indiana Academy: For each ripe banana, peel and place in blender. Cover with 15 percent NaCl solution, add two drops of dish soap, and blend to smithereens! Next, pour through several cheesecloth layers into a beaker. Add a pinch of meat tenderizer (contains enzymes) to the beaker to break down the proteins. Divide the solution in the beaker into test tubes (fill them halfway). Let the precipitate settle out (which takes 15 to 20 minutes). Use ice cold (put in freezer overnight) 95 percent ethanol. Layer the alcohol on top of the banana mixture and stir the area between the layers with a glass stirring rod or wooden splint. The DNA should stick to the stirring rod! Note that: - Blending breaks up the cells.
- The salt water denatures the proteins that could digest (lyse) the DNA.
- The banana is triploid.
- The soap acts as a surfactant to get the membranes, etc. out of the way."
-- Julie Smiley, Winchester Community High School, Winchester, Indiana. 2/23/00
Answer 6: "According to the Genetic Science Learning Center at the University of Utah, these detergents should work (for DNA extraction): Lemon Fresh Joy, Woolite, Ivory, Shaper, Arm & Hammer, Herbal Essence shower gel by Clairol, Tide, Dish Drops, Kool Wash, Cheer, Sunlight Dish Soap, Dawn, Delicate, All, and Ultra Dawn."
-- Bob Heun, Brooks School, North Andover, Massachusetts. 2/23/00
Answer 7: "There is a terrific and easy DNA extraction from wheat germ that I did even with an at-risk biology I class. The procedure is from the Hughes Undergraduate Biological Science Education Initiative. In a 50 mL test tube, add 20 mL hot water (50-60° C) to 1 gram raw wheat germ and rock it by hand for 3 minutes; then add 1 mL Lemon Fresh Joy or Woolite and rock gently every one-half minute for five minutes (to avoid foam -- remove any you form by pipette). Tilt the test tube and SLOWLY pour 14 mL 95 percent ethyl alcohol (rubbing alcohol can be used but precipitates out less DNA) so that it forms a layer on top of the wheat germ solution. After letting it sit for a few minutes, a stringy film of DNA should form at the interface."
-- Carolyn Schofield, Robert E. Lee High School, Tyler, Texas. 1/30/99
Tip: "The Web site "Amino Acids," from the University of Mississippi (see below), shows the amino acids with their respective letter designations. This might help reinforce primary structure of proteins in a fun way."
-- Carol Luterek, Maryvale High School, Cheektowaga, New York. 1/19/01
Multipart Question: "My AP Biology classes just finished our first try with PCR. We amplified the PV-92 loci for the alu sequence using the kit from Carolina. How do you handle the data within the context of the AP curriculum? Do you use the Web site to look at other populations and their Hardy Weinberg information?
Answer: "I would suggest that you use the DNA Learning Center's Web site because of all the lab extensions that can be done there."
"On some of the gels we got 'streaking,' many bands blurred, with the 550 and 850bp bands showing more prominently. Any ideas on how we can clear up the extra bands? The lab recommends a 'hot start.' Does anyone else do that and how do you handle it?"
Answer: "Keep everything on ice and bring the ice containers with samples to the thermocycler. Have the thermocycler prewarmed to 94° C. Load all samples as quickly as possible and start the program."
"If we finish the PCR one day and want to run the gels the next, do we need to store PCR products at 20 degrees or -4?"
Answer: "Store on ice in the refrigerator (0° C)."
"Finally, what is the latest word on the mutagenic properties of ethidium bromide? I was very cautious about student contact. However, I still worry about the risks."
Answer: "The EtBr used in this lab is an extremely weak solution. However, I recommend that the teacher is the only one to handle anything dealing with EtBr. The teacher should wear gloves and perform good laboratory practice. If possible, take pictures of the gels for the students to analyze."
-- Pat Ryan, Carolina Biological Supply Company, Burlington, North Carolina. 2/26/ 01
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