Lecture 2
DNA EXTRACTION
Isolation of DNA from Whole Blood using Puregene DNA Purification Kit
(Gentra Systems).
Procedure for DNA extraction
DNA will be extracted from whole blood using Puregene DNA Purification Kit
(Gentra Systems).
Principle
The PUREGENE DNA Purification Kit relies on biological or environmental
specimens as a source of genomic, mitochondrial or viral DNA. Typical sample types
include: whole blood, buffy coat, cultured cells, body fluids, animal or plant tissue,
and microbes.
Most mammalian whole blood and bone marrow specimens contain both nonnucleated cells (red blood cells) and nucleated cells (white blood cells) which contain
DNA. When purifying DNA from these whole blood or bone marrow specimens, the
red blood cells, which lack genomic DNA, are first lysed to facilitate their separation
from the white blood cells. DNA is purified from cells such as white blood cells,
animal tissue, cells contained in body fluids, or microbes, by first lysing the cells with
an anionic detergent in the presence of a DNA stabilizer. The DNA stabilizer works
by limiting the activity of DNases that are contained in the cell and elsewhere in the
environment. Contaminating RNA is then removed by treatment with an RNA
digesting enzyme. Other contaminants, such as proteins, are removed by salt
precipitation. Finally, the genomic DNA is recovered by precipitation with alcohol
and dissolved in a buffered solution containing a DNA stabilizer.
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Lecture 2
DNA purification from 10 ml whole blood
RBC Lysis
1)
10 ml whole blood will be added to a 50 ml centrifuge tube containing
30 ml RBC lysis solution. Invert to mix and incubat 10 minutes at
room temperature. The tube will be inverted again at least 20 times
during the incubation.
2)
The tube will be centrifuged at 3,000 RPM for 15 minutes. Supernatant
will be removed leaving behind the white cell pellet and about 200µl
of the residual liquid.
3)
The tube will be vortexed vigorously to re-suspend the cells in the
residual liquid. This will be greatly facilitated cell lysis in Step 4
below.
Cell Lysis
4)
10 ml Cell Lysis solution will be added to the re-suspended cells and
pipetted up and down to lyse the cells. If cell clumps are visible, tube
will be vortexed for 40 seconds and sample will be incubated at 37°C
until the solution is homogeneous. Samples are stable in cell lysis
solution for at least 18 months at room temperature.
Protein Precipitation
1)
2)
3.34 ml Protein precipitation solution will be added to the cell lysate.
The sample was vortexed at high speed for 50 seconds to mix the protein
precipitation solution uniformly with the cell lysate.
3)
The sample will be centrifuged at 3,000 RPM for 15 minutes. The precipitated
proteins shot formed a tight, dark brown pellet. If the protein pellet was not
tight, step 3 was repeated, followed by incubation at room temperature for 5
minutes and then step 4 was repeated.
DNA Precipitation
1)
The supernatant
will be poured, containing the DNA (leaving behind the
precipitated protein pellet), into a clean 50 ml centrifuge tube containing 11 ml
100% Isopropanol (2-propanol)
2)
The sample was mixed by inverting gently 20 times until the white
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Lecture 2
threads of DNA formed a visible clump.
3)
The sample will be centrifuged at 3,000 RPM for 3 minutes. The DNA was
visible as a small white pellet.
4)
Supernatant was poured off and tube drained briefly on clean absorbent paper.
10 ml 70% ethanol was added and tube inverted several times to wash the
DNA pellet.
5)
The sample will be centrifuged at 3,000 RPM for 1 minute. Carefully the
ethanol was poured off.
6)
The tube will be inverted and drained on clean absorbent paper and allowed to
air dry 20 minutes.
DNA Hydration
1)
2)
300 µl DNA hydration solution will be added.
DNA will be rehydrated by incubating at 65°C for 1 hour and vernight at
room temperature. Tube will be taped periodically to aid in dispersing the
DNA.
3)
Sample will be centrifuged briefly and then transferred to a 1.5 ml microfuge
tube.
4)
The purity and concentration will be checked for each DNA sample.
5)
From each sample 100 µl working DNA (50ng/ µl) will be labeled and stored at
4°C or – 20° C.
6)
Original stock of each sample of DNA will be labeled and stored at -80°C.
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Lecture 2
Spectrophotometer:
Nucleic acids, like many other substances, have the property of absorbing light at a
specific wavelength. DNA and RNA absorb light maximally at a wavelength of 260
nm. Because of this property, they can be quantified spectrophotometrically.
The purity of a nucleic acid solution can be determined by calculating the A260/A280
ratio.
The nucleic acid absorbs maximally at 260 nm and protein (a principle
contaminant) absorbs maximally at 280 nm.
Pure DNA has an A260/A280 ratio of 1.8.
Pure RNA has an A260/A280 ratio of 2.0.
If the ratios are significantly less than these values, then the sample is not pure.
1. The light source
Spectrophotometers have a tungsten lamp to make light with wavelengths in the
visible range (340 - 650 nm). Modern spectrophotometers also have a hydrogen lamp
which emits ultraviolet light (less than 340 nm). UV light is sometimes useful because
many biomolecules (especially proteins and DNA) absorb such low-wavelength light.
Using UV light we can detect nucleic acids and proteins without having to react them
with any colour-producing compounds!
5. Cuvettes
Solutions to be measured in a spectrophotometer are placed in a special container
called a cuvette. Cuvettes may be made of plastic, glass or quartz. Plastic cuvettes are
used for aqueous solutions, where the absorbance is measured in the visible range of
the spectrum. With organic solvents glass cuvettes should be used. The choice of
cuvette also depends on the wavelength at which the absorbance is to be measured.
Glass or plastic cuvettes can be used at wavelengths greater than 340 nm whereas
quartz cuvettes are used in the ultraviolet range. This is because plastic and glass
absorb UV light!
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Lecture 2
How to Use the Spectrophotometer
1. Prepare your blank as following:
98 µl (H2O) + 2 µl ( DNA Hydration Solution).
2. Prepare your samples as following:
98 µl ( H2O) + 2 µl ( your samples).
3. Turn on the Spectrophotometer.
4. Chose 50 as dilution factor.
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