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DNA ISOLATION
Learning Objectives
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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). The detergent, sodium laurel sulfate (SDS),
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 is dried. The DNA can then be dissolved in
water for further analysis or modification.
Notes:
1. Detergent action: In this figure, 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. Meat tenderizer contains enzymes that will strip away (digest) the histone proteins bound to
DNA. The two most common enzymes used in meat tenderizer are the proteases bromelain and
papain extracted from pineapple and papaya, respectively.
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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 also neutralizes the charges on the sugar- phosphate backbone of the DNA molecule.
Positively charged sodium ions neutralize the negative charge on the PO3- groups on the nucleic
acids, making the molecule less hydrophilic, and therefore less soluble in water. This enables the
DNA to be precipitated from solution 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:
 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 properly. 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.
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Reagents and equipment
Equipment
Cheesecloth + scissors
Model of DNA
Water bath at 60 degrees C
Blender
Digital balance
Weigh paper
50 ml conical tubes
graduated cylinder (10ml and 100 ml)
250 ml beakers
50 ml beakers
clean spatula
UV spectrophotometer and cuvettes
Centrifuge for 50 ml conical tubes
Mortar and pestle
Stir plate
Stir bar
Reagents
2 bottles of sterile dH20
Wheat germ (non-toasted)
NaCl (non-iodized) molecular weight 58.44 grams/mole
Fruit
Peas or onion
Detergent or 10% SDS
Ice-cold 95% Ethanol
70% Ethanol
Other
Ice in small ice bucket
Carboy of distilled water
Sharpie marker
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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. Whole wheat flour contains all parts of the wheat seed
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:
a. Add 90 ml distilled H2O to a 250 ml beaker
b. Obtain a piece of weigh paper. Make a crease in the weigh paper and then place on the
balance. Zero the balance.
c. Weigh 2 grams of NaCl (non-iodized salt) and place in the beaker of H20. Mix until the
salt is thoroughly dissolved.
d. Add 10 ml detergent (or 10% SDS) to the beaker. Mix.
*Record the final concentration of detergent in the DNA lysis solution.
*Record the final molarity of NaCl (molarity is expressed in moles per liter. The molecular
weight of NaCl is 58.44 grams/mole)
2. Weigh 2 grams 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 30 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 5 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.
*Record your observations
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II. Filter extract and precipitate DNA
1. Cut a double thick piece of cheesecloth and make a depression over a 50 ml beaker.
2. Filter the extract 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 extract.
* Record the volume of extract obtained.
*What is in the extract? (review all components in the homogenized tissue solution)
3. Pour 10 ml of the extract into a 50 ml conical tube.
4. Using a graduated cylinder, measure a 2X volume ice-cold 95% ethanol.
5. Gently slide the alcohol down the side of the beaker so that the alcohol forms a layer over the
extract. The DNA will precipitate at the interface. DO NOT MIX.
*Record observations. Include visual changes, especially at the interface. Be detailed. You may
include a sketch.
6. 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
*Record observations. Include a labeled sketch of the pellet and supernatant.
7. Gently rinse the pellet with 5 ml 70% EtOH. Repeat the centrifugation step if necessary.
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 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). X-ray
crystallography, the technique is the method used to produce a picture of the DNA molecule.
CLEAN UP
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Determination of DNA Concentration and Purity by UV Spectrophotometry
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A spectrophotometer is employed to measure the amount of light that a sample absorbs.
The instrument 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.
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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 chromogen (light absorbing molecule) in
solution and to the length of the light path through the solution.
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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
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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 ug/ml for double-stranded DNA
40 ug/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 single-stranded DNA molecule with an
absorbance reading of 0.62 at 260 nm.
1 OD
40ug/ml
=
0.62 OD
X ug/ml
X = 24.8 ug/ml
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In contrast to nucleic acids, proteins have a UV absorption maximum of 280 nm, due
mostly to the 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
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Procedure
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Obtain a cuvette. Do not touch the sides of the cuvette – no fingerprints.
Carefully add 100 ul of DNA sample
Add 900 ul of sterile H20. Use the pipetteman to mix gently by pipetting up and down.
Record the 260 and 280 readings
Results and Calculations
Tissue type ______________________
260 reading (OD 260) = ______________
=
_____________
ug/ml DNA
Multiply the concentration by the dilution factor. For example, if you diluted your sample by 4
(250 ul sample + 750 ul H20), multiply by 4.
Concentration of undiluted DNA sample
= ____________ ug/ml
Total DNA in test-tube
= ____________ ug DNA
(multiply ug/ml by total volume DNA)
See last lab's notes for the amount of water your entire DNA sample is in.
Total yield of DNA
(multiply the total DNA in test tube by the total volume of filtrate obtained) = _______ug DNA
Micrograms (ug) DNA obtained per gram tissue used
= ________ ug DNA per gram tissue
Calculating the purity of DNA
280 reading = _______________
260 /280 ratio = __________________
(divide the 260 reading by the 280 reading)
Comment on the purity of the DNA sample
Include these results and calculations in the laboratory manual
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Results and Calculations
Tissue type ______________________
260 reading (OD 260) = ______________
=
_____________
ug/ml DNA
Multiply the concentration by the dilution factor. For example, if you diluted your sample by 4
(250 ul sample + 750 ul H20), multiply by 4.
Concentration of undiluted DNA sample
= ____________ ug/ml
Total DNA in testtube
= ____________ ug DNA
(multiply ug/ml by total volume DNA)
See last lab's notes for the amount of water your entire DNA sample is in.
Total yield of DNA
(multiply the total DNA in test tube by the total volume of filtrate obtained) = _______ug DNA
Micrograms (ug) DNA obtained per gram tissue used
= ________ ug DNA per gram tissue
Calculating the purity of DNA
280 reading = _______________
260 /280 ratio = __________________
(divide the 260 reading by the 280 reading)
Comment on the purity of the DNA sample
Include these results and calculations in the laboratory manual