Laboratory 4b
Precipitating DNA out of Solution:
DNA Spooling
Background
DNA is arguably the most important molecule in living things. The long, thin fibers in the
molecule store all of the information needed to produce all of the molecules in an organism, either
directly or indirectly. The structure of a DNA molecule is related to its function, as it is with all
molecules.
To conduct genetic engineering, scientists need DNA in pure form. DNA must be purified
out of cells or viruses, isolating it away from other molecular contaminants, such as proteins,
carbohydrates, and lipids. Based on its molecular characteristics, DNA can be drawn out of
cellular or aqueous solution. These characteristics include the long double-helix shape and the
charged phosphate groups on the outer sugarphosphate backbone. The phosphate groups
are repelled by nonpolar solutions, such as
alcohol.
In this experiment, DNA molecules are
precipitated from solution and spooled onto
a glass rod (see Figure 4.2). The starting DNA
Hold the container and rod so the
stock solution contains DNA that has already
layers are parallel to the floor.
been isolated from the nuclei of salmon sperm
cells by technicians at the manufacturer.
They have collected sperm samples, exploded
the sperm cells, separated the contaminant
proteins from the DNA, pulled the DNA
out of the remaining aqueous solution using
EtOH
ethanol and then dried the pure DNA strands
bubbles trapped
to form the crystalline DNA purchased by your
in DNA strands
supervisor. Your supervisor has put the pure
aqueous
DNA into TE buffer and let it go back into
DNA layer
Figure 4.2. Spooling. Keep the glass rod almost parallel to the floor
solution. Your task is to pull the pure salmon
and scoop
at the
interface
between
when spooling so that Twirl
it is easier
to scoop
and
twirl DNA
molecules
testes DNA strands back out of solution for
theletalcohol
andstrands
aqueous
around the rod. Don’t
the DNA
fall(watery)
off the DNA
glass layer.
rod.
later use to provide the reagent DNA for this
experiment.
Purpose
Can DNA be spooled out of solution? What does it look like?
What yield of DNA can be recovered during the isolation?
Materials
Beakers, 50 mL
DNA, salmon testes, 4 mg/mL
Pipet, 2 mL
Pipet pump, blue
Micropipet, P-1000
Micropipet tips for P-1000
5M NaCl
Ethanol (EtOH), 95%
Glass rods
Tubes, 15 mL conical
Tube racks for 15-mL tubes
Permanent lab marker pens
Plastic beaker, 1L Tri pour®
Caution: Alcohol is
flammable. Keep away from
flame or ignition sources.
DNA Isolation and Analysis
69