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BIO 208
DNA ISOLATION
Learning Objectives

Demonstrate proficiency with the techniques involved in DNA extraction

Work with organisms composed of cells containing easily obtainable DNA

Learn in detail the role of specific reagents and equipment in the isolation of DNA

Analyze the yield and purity of DNA using UVspectrophotometry
Introduction to DNA isolation
In the DNA isolation procedure, cell walls (plants) and cell membranes are broken down by
tissue homogenization (via mashing or blending). Detergent solubilizes phospholipids in the cell
and nuclear membranes. Mashing, heat, and detergent facilitate cell lysis. A filtration step may
be included to remove solid components from those dissolved in the DNA lysis buffer. The
addition of alcohol precipitates the DNA, enabling DNA to be isolated from other solution
components. After the DNA has been separated by centrifugation, the alcohol is removed, and
the DNA dried. The DNA can then be dissolved in water for further analysis or modification.
1. Detergent action: Detergent disrupts the cell membrane phospholipids releasing membrane
proteins and liberating DNA into the solution.
2. DNA is highly soluble in water because the phosphate group of each nucleotide carries a
negative charge and associates electrostatically with polar water molecules. DNA is hydrophilic.
3. Papain enzyme can be used to strip away (digest) the histone proteins bound to DNA, thereby
facilitating isolation of pure DNA.
4.Endonucleases, also known as DNases and RNAses, are denatured at 60oC and thus
inactivated thereby protecting DNA from enzymatic degradation.
5. Salt (NaCl or NaOAc) assists in the denaturation and removal of histone proteins and exposes
DNA. Salt neutralizes charges on the backbone of the DNA molecule. Positively charged sodium
ions neutralize the negative charge on the PO3- groups, making the molecule less hydrophilic,
and less soluble in water. This enables DNA to be precipitated upon addition of alcohol.
6. DNA is not soluble in alcohol. When ethanol is added, the DNA precipitates at the
water/ethanol interface. Ethanol is the choice storage condition for DNA.
7. High molecular weight DNA is intact in long threads (chromosomes) To obtain high molecular
weight DNA:
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BIO 208



Wear gloves and do not introduce any items into the DNA which are not very clean
Do not touch or place pipettes or other items on lab bench
Do not vigorously shake DNA
High molecular weight DNA is best stored at 4oC. At -20oC, high molecular weight DNA may be
subject to extensive single and double strand breaks. -70oC is excellent for long-term storage
Centrifugation
Centrifugation separates components on the basis of particle size and density difference between
liquid and solid phases. Increasing the effective gravitational force on a test tube rapidly causes
solid or denser material to gather on the bottom of the tube. The supernatant, or liquid portion,
can be decanted from the tube without disturbing the pellet which is adhered to the lower portion
of the tube. The rotor must be carefully balanced by weight. A fixed angle rotor spun at 4,000
rpm is used in the DNA isolation procedure.
Observe the following rules for centrifugation
1. Never use an alkali on a rotor
2. Always clean and completely dry the rotor after use. Any spilled materials, especially
salts and corrosive solvents must be removed immediately with water.
3. Be especially careful not to scratch the surface of a rotor. Use plastic brushes only.
4. Always use the proper centrifuge tubes.
5. Always balance the rotor. Balance the tube with a medium that is identical to that being
centrifuged, i.e. do not balance an alcohol solution with water, or a dense sucrose solution
with water only -- the distribution of the densities will be incorrect.
6. Ensure that the rotor is properly seated within the centrifuge. Be sure the rotor cover is in
place and properly screwed down, where appropriate. NEVER use a rotor without its lid,
when one is supplied - the screw actually holds the rotor to the motor shaft.
7. Start the centrifuge and set the timer. Do not attempt to open the centrifuge until the rotor
has come to a complete stop.
Reagents and equipment
Cheesecloth + scissors
Model of DNA
Water bath at 60 degrees C
Digital balance
Weigh paper
50 ml conical tubes + rack
graduated cylinder (10ml and 100 ml)
250 ml beakers (2)
50 ml beakers (2)
clean spatula
Centrifuge for 50 ml conical tubes
Mortar and pestle
UV spectrophotometer and cuvettes
Reagents
bottle of sterile dH20
Wheat germ (non-toasted)
NaCl (non-iodized)
Fruit
Detergent or 10% SDS
Ice-cold 95% Ethanol
70% Ethanol
Carboy of distilled water
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BIO 208
Isolation of DNA from wheat germ or fruit
(Adapted from http://gslc.genetics.utah.edu/)
Wheat germ is obtained from wheat seeds. The germ is the embryo. When wheat seeds are
milled into white flour, the germ and bran (fiber) are removed, so that white flour consists of
starch.
The seed is the fruit of the strawberry
Record all observations in the laboratory notebook. Wear gloves to prevent contamination.
I. Homogenize and lyse cells and solubilize lipid
1. Prepare DNA lysis solution:
 Add 90 ml distilled H2O to a 250 ml beaker
 Obtain a piece of weigh paper. Make a crease in the weigh paper and then place on
the balance. Zero the balance.
 Weigh 2 grams of NaCl (non-iodized salt, molecular weight 58.44 grams/mole) and
place in the beaker of H20. Mix until the salt is thoroughly dissolved.
 Add 10 ml detergent to the beaker. Detergent is viscous! Mix gently to avoid bubbles.
2. Weigh 1 gram of raw wheat germ onto a piece of weigh paper. Place in a 250 ml beaker and
add 40 ml DNA lysis solution.
3.
Set up a separate beaker with 15 grams of fruit. Use a mortar and pestle to mash the fruit
thoroughly. Scrape into the beaker. Add 40 ml DNA lysis solution.
4. Label the beakers (initials) with a piece of tape and sharpie marker. Incubate in a 60oC water
bath for 10 minutes. Swirl the beaker every 2 minutes during the incubation period. Avoid
contaminating the experiment with water from the water bath.
5. Add 1 gram of meat tenderizer to each beaker, swirl and incubate at 60oC for 5 minutes.
Swirl the beaker every few minutes.
II. Filter extract and precipitate DNA
1. Cut 2 double thick piece of cheesecloth and make a depression over two 50 ml or 100 ml
beakers.
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BIO 208
2. Filter each extract (in separate beakers!) through the cheesecloth being careful not to let the
extract flow over the sides of the cheesecloth. Discard the cheesecloth and remaining
components. Save the filtered extracts.
3. Pour 10 ml of the extract into a 50 ml conical tube. Label the tube with your initials and
tissue type.
4. Using a graduated cylinder, measure a 2X volume ice-cold 95% ethanol.
5. Gently slide the alcohol down the side of each tube so that the alcohol forms a layer over the
extract. The DNA will precipitate at the interface. DO NOT MIX.
6. A. Wheat germ: centrifuge in a balanced centrifuge for 5 minutes at 4,000 rpm. Carefully
pour off the supernatant.
When two tubes are balanced, the level of fluid in
each tube is the same.
If one tube has less volume than the other, add
additional solution to that tube with a Pasteur pipette
B. Strawberry: Spool the DNA, drain the ethanol from the tube, and blot the tube on a paper
towel to remove all traces of ethanol. Add the spooled DNA back to the tube
7. Gently rinse the DNAs with 5 ml 70% EtOH. Centrifuge each tube for 5 minutes at 4,000
rpm. Decant the EtOH from the pellet. Invert the tube over a folded paper towel to remove
all traces of ethanol. Make sure the pellet does not slide out!
8. Air dry the pellet overnight or until ethanol has evaporated completely.
9. Add 10 ml sterile H2O and resuspend pellet by gently inverting the capped tube. Store at 4oC.
The double helix structure of DNA cannot be seen with the naked eye, or even with a
microscope. The width of the double helix is 1 billionth of a meter (1 X 10 – 9 meters).
CLEAN UP  wash all glassware and remove traces of soap.
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BIO 208
Determination of DNA Concentration and Purity by UV Spectrophotometry

A spectrophotometer is used to measure the amount of light that a sample absorbs. It operates
by passing a beam of light through a sample and measuring the intensity of light reaching a
detector. The beam of light consists of a stream of photons. When a photon encounters the
solution or molecule studied (in this case, DNA), there is a chance the molecule will absorb
the photon. This absorption reduces the number of photons in the beam of light, thereby
reducing the intensity of the light beam.

The Beer-Lambert law states that the amount of light absorbed is proportional to the number
of molecules of absorbing substance in the light path; i.e. absorption is proportional to the
concentration of the light absorbing molecule in solution and to the length of the light path
through the solution.

OD (optical density) measurements are made by comparing a solution containing the sample
to a reference solution. Usually, the reference is a blank, i.e., a solution identical in
composition with the sample except that the absorbing material being measured is absent
For pure solutions of DNA, the simplest method of quantitation is absorbance at a wavelength of
260 nm. A UV spectrophotometer must be used.
An optical density (OD) reading of 1 = a concentration of:
50 g/ml for double-stranded DNA
40 g/ml for single-stranded DNA and RNA
20-33 ug/ml for oligonucleotides (short single stranded pieces of DNA)
Example: Calculate the concentration in ug/ml of double-stranded DNA molecule with an
absorbance reading of 0.62 at 260 nm.
1 OD
50g/ml
=
0.62 OD
X g/ml
X= ?
Proteins have a UV absorption maximum of 280 nm, due mostly to tryptophan residues. The
absorbance of a DNA sample at 280 nm gives an estimate of the protein contamination of the
sample. The ratio of the absorbance at 260 nm/ absorbance at 280 nm is a measure of the purity
of a DNA sample; it should be between 1.65 and 1.85
Using an absorbance reading that falls closest to the middle of the sensitive range (range
is 0.1 – 1 OD) gives the most accurate results. The absorbance scale is logarithmic, so the
readings are more reliable at the low end of the scale. At the upper end of the range the
substance approaches saturation, so that there less resolution in absorbance readings.
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BIO 208
Prepare dilutions of DNA
1. Obtain a microfuge rack and 10 sterile microfuge tubes (1 set of 5 for each of the DNA
preparations, wheat germ and strawberry).
2. Label the tubes with the appropriate dilution
Dilution
1:2
1:5
1:10
1:20
1:50
µl DNA added
µl sterile water added
Total volume
1000 µl
1000 µl
1000 µl
1000 µl
1000 µl
3. Fill in the table. The total volume prepared for all tubes is 1 ml.
4. Prepare the dilutions using the wheat germ and strawberry DNA samples. Use a new pipette
tip each time being careful not to let the pipette tip touch anything but the DNA.
5. Cap tightly and vortex each tube
6. Read absorbance via spectrophotometry
7. Fill in the calculations in the results and calculations section of the laboratory notebook
Plot a standard curve
A standard curve is a quantitative research tool, a method of plotting data that is used to
determine the concentration of a substance, in this case, DNA. Spectrophotometry is performed
with known concentrations of DNA and the results plotted on a graph. The graph can then be
used to determine the concentration of an unknown DNA, based on the absorbance reading.
1.
2.
3.
4.
5.
Label each axis on the graph paper. Y axis = absorbance. X axis =DNA concentration ul/ml
Plot the following data
Draw the best fit straight line through the data points
Title the graph
Determine the concentration of the DNA sample using the standard curve
Absorbance
0.1
0.2
0.45
0.85
1
DNA concentration
5 ug/ml
8 ug/ml
22.5 ug/ml
42.5 ug/ml
50ug/ml
6. Does the concentration you plotted for the DNA samples agree with the concentration obtained from
using the formula
1 OD = 50 ug/ml DNA?
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BIO 208