Lab.

6
DNA extraction
from human
blood

Be introduced to the
laboratory techniques
involved in DNA extraction.
Test DNA integrity using
gel electrophoresis.
 DNA is used every day by scientists and
lawyers to help in criminal investigation,
paternity test, researches, . etc.
Your DNA is your genetic fingerprint, this
means that your DNA is like no one elses
in the world.
DNA is a nucleic acid, made of carbon,
hydrogen, oxygen, nitrogen, and
phosphorous.
 DNA can be considered the
hereditary code of life because
it possesses the information that
determines an organisms
characteristic and is transmitted
from one generation to the next.
You receive half of your genes from your mother and half from your
father. The more closely related organisms are, the more similar
their DNA.
Day to day, DNAs job is to direct the functioning within the cells of
your body.
 DNA is in the nucleus of almost every cell in
your body.
The length of DNA per cell is about 100,000
times as long as the cell itself. However, DNA
only takes up about 10% of the cells volume.
This is because DNA is specially packaged
through a series of events to fit easily in the
cells nucleus.
The structure of DNA, the double helix, is
wrapped around proteins, folded back onto
itself, and coiled into a compact chromosome.
 Individual chromosomes can be studied using
microscopes, but the double helix of a
chromosome is so thin that it only be
detected through innovative, high-tech
procedures.
Chromosomal DNA from a single cell is not
visible to the naked eye.
However, when chromosomal DNA is extracted
from multiple cells, the amassed quantity can
easily be seen and looks like strands of
mucous-like, translucent cotton.
 (Ethylenediamine tetracetic acid) is
known as a chelating agent, In other
words, it binds divalent cations such as
Mg and Ca.
This ion is used as a cofactor in nuclease
enzymes and must be made unavailable
to the cells if we want to end up with
nucleic acids as an end product.
 Acts as a buffer and raises
the pH of the solution in
preparation for the acids
added in the subsequent
steps of the DNA extraction
procedure.
 (Sodium Dodecyl Sulfate) is a
biological detergent which causes
the cell membrane to break down
further and emulsifies the lipids and
proteins of the cell by disrupting the
polar interactions that hold the cell
membrane together, and forms
complexes with these lipids and
proteins causing them to precipitate
out of the solution.
 (Sodium chloride) enables
nucleic acids to
precipitate out of an
alcohol solution because it
shields the negative
phosphate end of DNA
causing the strands to come
closer together and coalesce.
 DNA will be
precipitated by
adding cold alcohol
to the cell extract,
DNA will come out
of the suspension
and may be seen
and collected on a
glass rod.
 Basic Steps in Isolating
DNA from Clinical
Specimens
Separate WBCs from RBCs.
Lyse WBCs.
Denature/digest proteins.
Separate contaminants (e.g.,
proteins, heme) from DNA.
Precipitate DNA.
Resuspend DNA in final buffer.
Procedure
1. transfer 300l of blood to
900l of Cell Lysis Solution in
1.5ml microcentrifuge tube.
Important: Blood must be
collected in EDTA, heparin or
citrate anticoagulant tubes to
prevent clotting.
Eppendorf tubes
.Invert the tube 56 times to mix. 2
3. Incubate the mixture for 10
minutes at room
temperature (invert 23
times once during the
incubation) to lyse the red
blood cells.
4. Centrifuge at 13,000g for
20 seconds.
Centrifuge
5. Remove as much supernatant as
possible without disturbing the visible
white pellet. Approximately 1020l of
residual liquid will remain in the 1.5ml tube.
6.Vortex the tube vigorously until
the white blood cells are
resuspended (1015 seconds).
7. Add 300l of Nuclei Lysis
Solution. Pipet the solution 56
times to lyse the white blood cells.
8. Add 100l of Protein
Precipitation Solution to the
nuclear lysate, and vortex
vigorously for 1020 seconds.
9.Centrifuge at 13,000g for 3
minutes.
10. transfer the supernatant to a
clean 1.5ml microcentrifuge tube
containing 300l of isopropanol.
11. Gently mix the solution by
inversion until the white
thread-like strands of DNA form
a visible mass.
Centrifuge at 13,000g for 1. 12
13. Decant the supernatant,
.minute
and add 300l of 70%
ethanol to the DNA.
14. Gently invert the tube several
times to wash the DNA pellet.

Centrifuge at 13,000g for 1. 15

.minute
16. Decant the ethanol and
invert the tube on clean
absorbent paper and air-dry
the pellet for 1015 minutes.
16. Add 100l of DNA
Rehydration Solution to the
tube and rehydrate the DNA.
Incubating the solution at. 17
65C for 1 hour or
.overnight at 4C
Freeze at -20 or
-70C for long-term
. storage
 Gel electrophoresis:
Prepare 1% a garose gel and mix
10 l DNA samples with loading dye
buffer as described in Lab 3,
Transfer the DNA samples into the
wells of the gel using a pipette.
Loading DNA samples must be done
slowly and smoothly to prevent
sample from squirting up into the
running buffer and diffusing away.