Explorations in DNA Extraction
Anything that is or was living has to have DNA in it. Hence, you should be able to extract DNA from
a great variety of sources. There are a
few basic steps to the process.
- A
sample. Examples include green
split peas or dry lima beans, frozen spinach, chicken liver, onions,
broccoli, raw wheat germ, kiwi fruit, bananas, banana chips soaked overnight in water, strawberries, yeast, calf
thymus, dog testes, frozen worms.
How much? Start with a
measured amount (a one-inch chunk, or one tsp or 10 grams or ½ cup.) Then if you do not see DNA at the end,
use more of the sample and less water.
- Add
water (about twice as much as your sample volume) – to let the DNA go into
solution. Many protocols call for
hot water.
- Add
large pinch of table salt – about
¼ tsp or 1.5 g.
- Add detergent – preferably a clear
detergent – dishwashing liquid. How much?
About equal to the amount of water you started with – if you used
about 50 ml of water, add about 50 ml of dishwashing liquid.
- Mix
with stirring or use a blender. At
this point you want to have a thin opaque solution (you can’t see through
it). Allow 5-10 minutes for salt
and detergent to work to break down the cells’ wall and membrane. Putting the sample container into a hot
water bath may help at this point.
Some protocols recommend heating at this point. (about
50oC.)
- Filter
to remove the cell debris – you can use cheesecloth or coffee filter. Or you can spin in centrifuge. You want to discard the solid material
and keep the filtrate (or supernatant from centrifugation). Put the filtrate (supernatant) into a
test tube.
- Add a
pinch of enzymes that break
down proteins – sources include Adolph’s meat tenderizer (use a pinch/5 g.)
or 6% papain (1 ml) or contact lens cleaner with
papain (1-2 mL) or
juice of fresh papaya or fresh pineapple (canned juice will not work). These enzymes break down the proteins
clinging to the DNA and also the proteins that break down the DNA. Stir
gently.
- Tilt
your test tube and slowly drizzle cold
alcohol (70% ethanol or isopropyl alcohol) down the side of the test
tube – use about the same amount of alcohol as filtrate (if you have about
5 mL liquid, add 5 mL
alcohol). Let the alcohol form a
layer on top of your liquid. Do not mix! Let sit till you see DNA.
Now you should see DNA rising from the liquid on bottom into
the alcohol layer at the interface. It
has been described as slimy or stringy or fluffy, like mucus, like “snot.”
Troubleshooting:
if you don’t see DNA, maybe your sample had a lot of water – so try
increasing the amount of sample you use.
Make sure your blended sample is opaque.
For Further Research:
try one of these ideas or some of your own:
- Experiment
with other DNA sources. Which
source gives the most DNA? How can
you compare?
- Experiment
with different soaps and detergents:
powdered versus liquid, shampoo?
- Experiment
with leaving out or changing steps.
See if that makes a difference in your DNA yield.
- Some
protocols use a buffer to maintain a proper pH – our protocol did not use
a buffer. A simple buffer recipe: use 50 g. sugar, 3 g. Epsom salts, one
buffered aspirin, add water to 500 mL. See if using a buffer instead of water
(in step 2) improves your results.
If you do further experiments that compare results, you want
to design a controlled experiment. You
would use precise measurements and keep a detailed record of your procedures
and results.
- You
would use graduated cylinders to record volumes and a balance to record
weights.
- You
would record how much time you allowed for each step for mixing and
blending and incubation.
- You
would want to “control the other variables” – that is, do every step
exactly the same except for the one variable you are testing – like for
example, comparing papaya juice versus meat tenderizer as an enzyme source.
- You would
record each step of your procedure fully and carefully so that someone
else could read your record and exactly duplicate your experiment.
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Trial 1
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Trial 2
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Trial 3
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Trial 4
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Sample – source/amt
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Water/buffer - volume
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Salt – measure
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Detergent – source/amt
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Mix time
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Filter/spin step
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Enzyme – source/amt
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Alcohol – source/amt
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DNA RESULT
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