Molecular Biology
MLAB 3102 (Practical )
DNA Extraction and Purification
Dr. Alaa Saud Alhegaili
DNA extraction
DNA extraction is a routine procedure to isolate &
collect DNA.
DNA extraction is the first step for subsequent
molecular analysis.
DNA can be extracted from almost any intact cellular
tissue
The product of DNA extracted will be used in
subsequent experiments
Poor quality DNA will not perform well in PCR
The principle of DNA extraction depends on the same
basic steps but there are some different methods details
depends on the type of sample and any substances
Biological sources used for DNA isolation
Blood ( EDTA top tube)
Cigarette Butts
Semen Envelope & Stamps
Saliva Fingernail Clippings
Urine Chewing Gum
Hair (w/Root & Shaft)
Bite Marks
Teeth Feces
Bone
Tissue
Laboratory precaution in handling
DNA samples
Handling fresh and stored material before extraction of DNA
Use either fresh samples or samples that have been quickly frozen in
liquid nitrogen and stored at –70°C.
This procedure minimizes degradation of the DNA by limiting the activity of
endogenous nucleases. (what is the function of this enzyme??)
For Blood Sample:
use fresh blood or
blood stored for < 2 days at room temperature.
Blood stored for 7 days at 2-8°C or for < 1 month at –15 to –25°C will result
in a 10 to 15% reduction of yield of genomic DNA.
Collect blood samples in tubes containing EDTA as anticoagulant, not
heparin.
Heparin can cause attenuation or inhibition of amplification during PCR.
However, if heparin cannot be avoided, the High Pure PCR Template
Preparation Kit can be used to remove the heparin from the sample.
Pipetting DNA
Avoid vigorous pipetting.
Pipetting genomic DNA through small tip openings
causes shearing or nicking.
Use tips with wide openings, specially designed for
genomic DNA.
Regular pipette tips pose no problem for plasmid DNA
and other small DNA molecules
Storage of DNA
Store genomic DNA at 2-8°C, storage at –15 to –25°C
can cause shearing of the DNA.
Plasmid DNA and other small DNA molecules can be
stored at 2-8°C for short term storage, or in aliquots at
–15 to –25°C for long term storage.
Store modified DNA at 2-8°C.
Manipulation of DNA
Always keep the DNA sample on ice when preparing an
experiment.
Drying DNA
Avoid over-drying of genomic DNA after ethanol
precipitation.
Let the DNA air dry.
Plasmid DNA and other small DNA molecules can be air or
vacuum dried.
Safety precautions
The handling of blood samples should be
carried out with great care, and they should all
be treated as potential sources of infection.
Handling and disposal of the various reagents
must be assessed and addressed properly in the
local laboratory standard operating procedure.
Dissolving DNA
Dissolve DNA in Tris buffer (e.g., 10 mM Tris, pH 7.0 –
pH 8.0).
To help dissolve the DNA, carefully invert the tube
several times after
adding buffer and/or tap the tube gently on the side.
Alternatively, let the DNA stand in buffer overnight at 2-
8°C.
Do not vortex genomic DNA.
Heat DNA for 10 min. at 65°C to dissolve and inactivate
DNases.
LAB EQUIPMENT (to be used in this experiment)
1. Automated pipettes
2. Micro-centrifuge
3. Water bath
4. UV-spectrophotometer
DNA extraction principle
1. Disruption or Lysis of nucleated cells
2. Removal of contaminants ( e.g. membrane lipids,
cellular and histone proteins)
• Lipid removed by detergents / chemicals
• Proteins bound to the DNA removed by a- adding a protease,
phenol/chloroform, salt (e.g. sodium or ammonium acetate)
3. Precipitation of DNA by using alcohol (e.g.
ethanol or isopropanol)
4. Re-suspend DNA in aqueous solution
5. Measurement of the UV absorbance at 260/280
ratio
6. Calculate the DNA concentration
Phenol and/or chloroform DNA extraction
The extraction of nucleic acids involves adding an
equal volume of phenol-chloroform to an aqueous
solution of lysed cells or homogenized tissue, mixing
the two phases, and allowing the phases to separate by
centrifugation
Centrifugation of the mixture yields two phases: the
lower organic phase and the upper aqueous phase.
Phenol and/or chloroform DNA extraction
Principle of Phenol and/or chloroform DNA extraction
1. Cell Lysis Buffer : lyses outer cell membrane, nuclei are
intact by using Ionic detergent, Salt, Buffer, EDTA: designed
to lyse outer cell membrane of blood and epithelial cells, but
will not break down nuclear membrane
2. Add Phenol/ Chloroform: The standard way to remove
proteins from nucleic acids solutions is to extract once with
phenol, once with a 1:1 mixture of phenol and chloroform,
and once with chloroform. This results into DNA released
into solution
3. Alcohol Precipitation of DNA: add 95% ice cold ethanol
and salt
4. Re suspending DNA: Precipitated DNA is washed with 70%
ethanol, dried under vacuum and re-suspended in TE buffer.
Advantage and disadvantages
Advantage Disadvantage
• yields relatively pure, high • Time consuming
molecular weight DNA • Requires sample to be
• DNA is double stranded – transferred to multiple tubes
good for PCR – increases risk of
contamination
• Involves use of hazardous
(and smelly!) chemicals
DNA measurement methods
• Different methods for assessing quantity &
quality of extracted DNA
• Absorbance (optical density)
• Agarose gel electrophoresis
• Use of fluorescent DNA-binding dyes.
• All three methods are convenient, but have
varying requirements in terms of equipment
needed, ease of use, and calculations to
consider.
DNA Measurement by spectrophotometer
DNA concentration can be calculated with using the optical density
that is measured by spectrophotometer
Measurement of the amount of UV irradiation absorbed by the DNA
bases is the principle of indicating the DNA amount
spectrophotometry method for quantifying DNA is better if sample is
pure (i.e. without significant amounts of contaminants such as a
proteins, phenol, agarose, or other nucleic acids)
Equipment used for the measurement
• A spectrophotometer equipped with a UV lamp,
• UV-transparent cuvettes (depending on the instrument) and
• A solution of purified DNA
DNA Measurement by spectrophotometer
DNA concentration is estimated by measuring the
absorbance at 260nm (A260) where DNA absorbs
light most strongly),
if sample is not clear pure , adjust the A 260
measurement for turbidity (measured by
absorbance at 320nm), multiplying by the dilution
factor, and using the relationship that an A 260 of 1.0
= 50µg/ml pure dsDNA.
The A260 reading range (generally 0.1–1.0).
• The reading at 260 nm allows calculation of the
concentration of nucleic acid in the sample.
• 1 O.D. at 260 nm for double-stranded DNA = 50
ng/ul of dsDNA
• 1 O.D. at 260 nm for single-stranded DNA = 20-
33 ng/ul of ssDNA
• 1 O.D. at 260 nm for RNA molecules = 40 ng/ul
of RNA
• The reading at 280 nm gives the amount of
protein in the sample.
DNA concentration
Concentration (µg/ml) = (A260 reading – A320 reading)
× dilution factor × 50µg/ml
• Total yield is obtained by multiplying the DNA
concentration by the final total purified sample
volume.
• DNA yield (µg) = DNA concentration × total sample
volume (ml)
Disadvantage of A260
DNA is not the only molecule that can absorb UV light at
260 nm.
Since RNA also has a great absorbance at 260 nm, and the
aromatic amino acids present in protein absorb at 280 nm,
both contaminants, if present in the DNA solution, will
contribute to the total measurement at 260 nm.
Additionally, the presence of guanidine will lead to higher
260 nm absorbance. This means that if the A260 number is
used for calculation of yield, the DNA quantity may be
overestimated.
DNA purification OD260/OD280 ratio
The most common purity calculation for DNA purity is the ratio of the
absorbance at 260 nm divided by the reading at 280 nm.
Good-quality DNA will have an A260/A280 ratio of 1.7–2.0.
A reading of 1.6 does not render the DNA unsuitable for any
application, but lower ratios indicate more contaminants are present
e.g. protein or phenol
The ratio can be calculated after correcting for turbidity (absorbance
at 320 nm).
• DNA purity (A260/A280) = (A260 reading – A320 reading) ÷
(A280 reading – A320 reading)
• ter cuvette. The DNA concentration read will
then be:
• OD260 * 50 ng/ul * dilution factor
• For example, if have OD260 = 1.6. Then the
concentration is:
• 1.6 * 50 ng/ul * 100 = 8000 ng/ul or 8 ug/ul
Storage Conditions
• Store DNA in TE buffer at 4 °C for weeks or
at –20 °C to –80 °C for long term.
<4 Months 1–3 Years<7 Years >7 Years
2–25 °C 2–8 °C –20 °C –70 °C
Recommended
for long-term
storage in
ethanol
