| Materials:
-Calf Thymus
(also called sweetbreads) may be cut into pieces and frozen for
future use.
-Isotonic
sucrose solution ( 106.1 grams table sugar in 1 liter of water
)
-Blender
-Cheese
cloth
-Funnel
-Detergent
solution ( 100 mL dishwashing liquid [ I've used Dawn], 15 grams
NaCl [table salt works fine]: add distilled water to make a final
volume of 1000 mL) ethanol (95%) dispensed into 3-5 mL amounts
into screw top test tubes or small containers and stored in a
freezer.
-One container per student group.
-2 test tubes per student group
- inoculating
loops or thin wires bent to form loop at the end for drawing up
DNA from test tube
As much as possible, you would like to work with cold materials
and solutions. When the alcohol is removed from the freezer it
should be carried on ice into the classroom.
Procedure
To break open
the thymus cells: Add a small piece of thymus (slightly smaller
than an egg) to the blender with enough sucrose solution to make
a thin milkshake consistancy. Blend until thymus is broken up.
Kids think this is cool and gross to watch!
Pour the blended
thymus through a funnel which is lined with four thicknesses of
cheese cloth. The filtrate collected below the funnel will contain
many nuclei which can be observed under the microscope. This step
removes gristle and other stuff.
Pour about 2
mL of the nuclei suspension collected from step 2 into a clean
test tube for each student group.
Pour about 2
mL of detergent solution into another clean test tube for each
group.
Students pour
detergent solution into test tube of nuclei suspension and gently
shake tube from side to side to mix. Do not shake enough to get
foamy! In this step the detergent will break open the lipid membrane
of the nuclei and release DNA. The salt in the solution will help
neutralize the negative charges on the DNA molecules so they will
precipitate.
Slowly pour COLD
ethanol down the inside of the test tube containing the nuclei/detergent
mixture so that a layer of ethanol forms on top of the mixture.
DNA will come out of solution at the place where the two layers
meet.
Use clean wires
to dip into layer where the two solutions meet and stir gently.
Students will be able to lift out masses of DNA. I have had students
pull gently until they lifted about 2 meters of long chain molecules
out.
For more information
visit: http://www.pbs.org/wgbh/aso/tryit/dna/replication.html
original source: http://www.accessexcellence.org/AE/ATG/data/released/0338-MaryJoOsborne/index.html
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