Download DNA Isolation: plant materials

Lab #___: DNA Isolation
Name(s):___________________________________________________
Date:
__________
Introduction:
The purpose of this lab is to give students the opportunity to extract, observe,
and analyze DNA. These are all fundamental skills involved in Biotechnology.
The DNA in a cell is about 100,000 times as long as the cell itself. However,
DNA only takes up about 10% of the cell's volume. This is because the highly
convoluted (folded) DNA molecules are packed into chromosomes in the cell's
nucleus.
The walls of plant cells are made of cellulose, which is a polysaccharide
comprised of glucose units. Cellulose provides a tough barrier that protects the cell.
In order to examine the contents of a plant cell, you need a physical means, such as
blending, to burst open the cell's walls and reveal the contents.
Plant cells also have membranes that envelop their contents. Cell membranes
are composed of a double layer of lipids that allow the movement and transfer of
certain ions and substances in and out of the cell. Breaking down a cell's membrane
allows you examine the contents of the cell. Detergents, such as soap, contain
chemical compounds that can break down cell membranes. Examples of these
compounds include SDS (sodium dodecyl sulfate) and lauryl sulfate.
Procedure
1. Put on the lab apron, safety goggles, and heat-resistant
waterproof gloves. Use the knife to coarsely chop the onion.
Place the chopped onion in the blender.
Tip: Do not spend a lot of time cutting the onion into very
small pieces before liquefying it in the blender. Let the
blender do the work.
2. Using the 50-mL conical tube as a measuring device, add 60 mL water to the
blender.
3. Place the lid on the blender and make sure that it is secure. Blend at low for
30 seconds to start macerating the onion. Blend at liquefy for 10 seconds,
pause, check, and agitate. Repeat 2 times or until the onion is thoroughly
liquefied.
4. Transfer 7 mL of the liquefied onion to a 15-mL tube. Pouring onion sludge
into the small opening of the tube is tricky. Use a wide funnel or roll up a
piece of paper to facilitate pouring. Fill the tube until it is about half full.
Tip: pour the liquefied onion from the blender into a beaker and then pour it
from the beaker into the 15-mL tube. This is much easier than trying to pour it
directly from the blender into the tube. Indicate pouring levels on your tubes
(50 mL and 15 mL) with a dark line.
5. Add 7 mL cell lysis solution to the tube. When this is added,
the 15-mL tube should be nearly full. Cap the tube.
6. Invert the tube to mix the contents, but do not shake it.
7. Place the tube in a water bath at 60 to 65° C for 15 minutes.
Tip: Use some type of tube holder in the water bath (for the 15-minute
incubation at 65° C) to keep the tubes in place.
8. Remove the tube from the water bath.
9. Place a conical filter into the mouth of the 50-mL tube and slowly pour the
contents of the 15-mL tube into the filter. Let the solution drip for awhile. If
necessary, squeeze the liquid through, but do not allow solids to fall into the
50-mL tube.
Tip: Place the lysate in an unobstructed area as it drips into the flatbottomed 50-mL tube.
10. Ensure that the DNA precipitation solution (95% ethanol) is
ice-cold when it is poured onto the lysate, i.e., the "pink stuff"
containing onion DNA. Hold the tube containing the lysate at
a 45° angle and slowly allow the precipitation solution to slide
down the surface of the tube to form a nice layer on top of
the lysate. There should be fairly equal amounts of clear
liquid (DNA precipitation solution, consisting mainly of
ethanol) and pink liquid (DNA lysing solution, consisting of
onion DNA, soap, and salt).
Tip: Slowly pour the chilled ethanol down the side of the
tube so as to prevent the 2 liquids from mixing.
11. Observe the DNA as a cloud of fine, viscous threads where
the 2 liquids meet. This is called the interface.
12. Insert the wooden splint into the 50-mL tube until its tip is just
below the interface. Turn and twist the splint, gently passing
it back and forth between the 2 layers of liquid. The DNA
should wrap around the splint like yarn on a spool.
Student Questions:
1. Explain why you carried out each of the 12 steps in the procedure.
2. What other foods could be used in this lab?
3. How pure is the DNA that you isolated?