Is It DNA?
When students "spool" DNA, they are told that the white stuff is DNA. But there is no "proof" that
it is DNA...
Ask students if they can think of ways to prove that the white spooled stuff is DNA.
- analyze composition chemically for A, T, C and G's and sugar-phosphate backbone
- use to change an organism's genotype (and phenotype?)
One way to "prove" a substance is DNA, albeit still indirectly, is to mix DNA with a chemical
that reacts with DNA specifically. The chemical we choose to use is ETHIDIUM BROMIDE, because
students will use that chemical in a subsequent lab to visualize the DNA samples on an agarose gel.
Give them the information that when ethidium bromide is mixed with DNA in solution
(not precipitated DNA), the dye slips into the double helix between the stacking bases.
In that location, the ethidium bromide, when exposed to ultraviolet light, fluoresces
an orangey-pink color.
Show students that you have a UV transilluminator as a source of ultraviolet light,
and you have a tube of ethidium bromide (concentration is 5 µg/mL, which is
10X more concentrated than we use to stain gels, SO BE CAREFUL). Invite students to help you design an experiment, using ethidium bromide as an identifier of DNA, to "prove" that what they spooled out of solution is DNA, and not, say protein.
OUR SUGGESTED METHOD:
- Identify what needs to be tested:
The spooled DNA. The DNA will have to be resuspended in solution in order for the ethidium bromide to slide into the helix. Placer spooled DNA into a sterile test tube with 1 mL of TE buffer (10 mM Tris, 1 mM EDTA, pH 8 - in the kit) and let resuspend overnight (or maybe sooner - watch to see if some of it redissolves during the class period).
- Discuss controls:
(1) Positive control: known DNA sample (for us, the starting solution of Salmon Sperm DNA).
(2) Negative control: water (or TE buffer)
(3) Negative control: another substance that could be co-purified with DNA from cells (protein, RNA). We suggest protein: egg white.
Ideally, you could do this as a demo for the whole class at one time if you had the MiniVisionary set up. Else, teams of students could file by the laid out experiment.
- Place a piece of clear plastic wrap, plastic baggie, transparency on the surface of the UV monitor (to protect the surface and for easier clean-up).
- Wearing gloves and goggles, pipet FOUR samples of 10 µL of ethidium bromide (5 µg/mL)
in a row on the plastic wrap.
- Into the first 10 µL "drop", mix 10 µL water or TE buffer.
- Into the second 10 µL "drop", mix 10 µL egg white.
- Into the third 10 µL "drop", mix 10 µL control DNA.
- Into the fourth 10 µL "drop", mix 10 µL resuspended DNA.
Ask students what they would expect to see in each drop (fluorescence or no fluorescence).
Turn on the UV transilluminator (and put the camera in place if using the MiniVisonary - ratchet up the exposure until the drops are visible on the monitor). Voila.
- Drop 1 (water or TE buffer): no fluorescence
- Drop 2 (egg white = protein): no or very little fluorescence
- Drop 3 (control DNA = original sample?): bright fluorescence
- Drop 4 (spooled DNA, resuspended in TE buffer): bright fluorescence
Day of spooling: TE buffer, sterile 15 mL tube, P-1000, sterile blue tips
Day of test: UV transilluminator, MiniVisionary (opt.), EtBr (5ug/mL), P-20, sterile yellow tips, plastic wrap (or substitute), TE buffer, egg white, salmon sperm DNA, gloves, goggles, student samples of resuspended DNA.