Spring 06

Plant physiology 347

Laboratory Block 3: Plant DNA extraction
Introduction
Deoxyribonucleic acid (DNA) is a nucleic acid usually in the form of a double helix that
contains the genetic instructions specifying the biological development of all cellular forms of
life, and most viruses. DNA is a long polymer of nucleotides and encodes the sequence of the
amino acid residues in proteins using the genetic code, a triplet code of nucleotides. In complex
eukaryotic cells such as those from plants, animals, fungi and protists, most of the DNA is
located in the cell nucleus. By contrast, in simpler cells called prokaryotes, including the
eubacteria and archaea, DNA is not separated from the cytoplasm by a nuclear envelope. The
cellular organelles known as chloroplasts and mitochondria also carry DNA.
The process of isolating DNA from cells and/or tissue is the first step for many laboratory
experiments in genetics. This process is also sometimes referred to as a DNA extraction
procedure. The goal of this process is to isolate or separate the DNA from the rest of the
components of the cell so that the DNA may be used for further downstream experiments.
For the majority of experiments, DNA is isolated in fairly small quantities. We will be isolating
genomic DNA from fresh cow liver tissue which will allow you to see the DNA

Figure 1: The structure of DNA

Even though the directions or protocol is given to you below you should carefully read
through this and be sure you understand what is occurring at each step. How is the DNA being
separated from the other cellular components? What role does centrifugation play? What role
does the NaCl solution play and what is the role of the ethanol?

Spring 06

Isolation and Quantification of Genomic DNA

Learning objectives for this lab:
Understand the basic process behind a DNA isolation (extraction) procedure
To get a feel for what DNA looks like
To learn how to determine the concentration of DNA using spectrophotometry
To learn how to make a standard curve
First: Read through the entire lab. Focus on the questions you will need to answer.
Second: With your group, sketch out a flow chart of each step. Each person should have this in
their notebook. Use diagrams/short sentences.

DNA Quantification
You will also learn how to determine the concentration of the DNA you isolate using a
spectrophotometer. You will measure the absorbance of the DNA solution you isolate and
compare it to a DNA solution of known concentration.
DNA Isolation Protocol
1. Weigh out 60 g of onion and mince it into smaller pieces using a razor blade (carefully!). You
may use the scale at the back bench to check the weight of your pieces.
2. Put your minced onion and 50 ml of the cold citrate-saline buffer in the blender and blend on
high for 30 seconds then low speed for 1 min 2min. You want to ensure that there are no large
chunks left.
3. Place 4 layers of cheesecloth over the top of a beaker. Pour the homogenized onion through
the cheesecloth and into the beaker. You may have to pour a little, let it filter through and then
pour more. You may also have to squeeze the homogenate through the beaker with your hand.
4. Add your filtered homogenate to a 50 ml centrifuge tube. If the homogenate is foamy, use a
pipette to remove the foam and add the rest of the liquid homogenate. Use a wax pencil to mark
the top level of the liquid in your tube and to identify it as belonging to your group.
5. Each group should now have a tube of filtered homogenate and your instructor will help
ensure that all the tubes are balanced and load them into the refrigerated centrifuge. The tubes
will be centrifuged at 6000 rpm for 10 min.
6. Remove your tube from the centrifuge being careful not to disturb the pellet. Gently pour off
and discard the supernatant. Add fresh, cold citrate-saline half-way (ie. use half the amount this
time) to the mark you made on the tube. Use a pipette to break up the pellet, you may also cover
the top of the tube with a lid and shake or vortex until all of the pellet is resuspended.
7. Centrifuge again at 6000 rpm for 10 min.

Spring 06
8. Again, discard the supernatant. This time, resuspend the pellet in 50 ml (use the mark on your
tube) 2.6 M NaCl solution, using same method as in #6.
9. Centrifuge again at 6000 rpm for 10 min.
10. This time pour the supernatant into a 250 ml beaker that has been sitting on ice. At this
point, the supernatant contains the DNA in your solution. You will discard the pellet. KEEP
THE DNA SOLUTION CHILLLED.
11. Add an equal volume (you can estimate) of ice cold ethanol to the lysate but you must do it
by SLOWLY pouring the ethanol down the side of the beaker. The goal is to form a layer of
ethanol on top of the DNA solution. The DNA will precipitate at the interface between the
alcohol and DNA solution.
12. Using a glass stirring rod, stir the precipitate the forms at the interface. Try to wind the
precipitate onto the rod until you get a fairly big blob. Use the stirring rod to remove the blob
and then gently let most of the alcohol drip off onto a kimwipe. You can also try to gently blot
the DNA on the kimwipe. The objective is to get as much alcohol off of the DNA blob as
possible.
13. Transfer the DNA to a clean, regular-sized test tube containing 10 ml of distilled water and
gently remove the DNA from the rod by agitating the rod up and down in the water. Cover the
tube and shake it until all of the DNA goes into solution (ie. you cant see it anymore). This is
referred to as the resuspension step.
Determining the concentration of the DNA solution
A spectrophotometer measures the amount of light, at a given wavelength, that is able to pass
through a solution. In other words, it measures the amount of light that a solution will absorb at
a given wavelength. You will be adding something called Dische reagent to your DNA solution,
which stains the DNA and causes a color change. The stronger the color change, the more
DNA present and hence the more absorbance of light that will occur. We will be using a
wavelength of 500 nm which is optimal for the Dische reagent. According to Beers Law, there
is a direct relationship between the concentration of a solution and the absorbance. This
relationship is: A = kc, where A=absorbance, k = constant of the instrument, and c =
concentration.
You will be blanking the spec. using Dische reagent alone. The blank should represent a
concentration of DNA that = 0 and therefore would have an absorbance = 0 or, transmittance =
100%.
Protocol:
1. Label 6 regular test tubes from 1 through 6 with a wax pencil.
2. Put 2 ml of distilled water in tubes 2 through 5. Leave tube 6 empty for now.

Spring 06
3. Place 2 ml of the standard DNA solution at 1 mg/ml (given to you) in tubes 1 and 2. Gently
mix the contents of each tube. Set tube 1 aside.
4. Transfer 2 ml from tube 2 into tube 3. Mix tube 3 well.
5. Transfer 2 ml from tube 3 into tube 4. Mix tube 4 well then discard 2 ml of tube 4.
6. Add 2 ml of your onion DNA solution to tube 6. You should now have 2 ml of solution in
each tube 1-6.
7. Add 4 ml of Dische reagent to each of the 6 tubes. Mix well and cap the tubes with aluminum
foil to prevent evaporation.
8. Heat the tubes in the boiling water bath for 10 min.
9. Cool the tubes on ice for a few minutes.
10. Pour the contents of each tube into the smaller tubes used in the spec. Use tube 5 to blank
your spec. which you should have set at 500 nm.
11. Read the absorbance of each of the other tubes filling in the data on the report sheet you will
hand in next week. You should also record this info in your lab notebook.
12. Using standard graph paper, plot the absorbance of tubes 1 through 4 on the Y axis and the
DNA concentration (mg/ml) of these same tubes on the X axis. You can determine the DNA
concentration because you started with adding 2 ml of 1 mg/ml standard DNA solution to tube 1
and 2 ml of 1 mg/ml solution to 2 ml of water in tube 2 You then made dilutions of this in tubes
3 and 4.
13. Label your remaining 6 ml of cow DNA solution and give to your instructor.