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 else’s
in the world.
• DNA is a nucleic acid, made of carbon,
hydrogen, oxygen, nitrogen, and
phosphorous.

DNA’s job is to direct the functioning within the cells of your body. the more similar their DNA. .• DNA can be considered the hereditary “code of life” because it possesses the information that determines an organism’s characteristic and is transmitted from one generation to the next. • You receive half of your genes from your mother and half from your father. • Day to day. The more closely related organisms are.

and coiled into a compact chromosome. is wrapped around proteins. DNA only takes up about 10% of the cell’s volume. • This is because DNA is specially packaged through a series of events to fit easily in the cell’s nucleus. • The length of DNA per cell is about 100. folded back onto itself. the double helix. . However.• DNA is in the nucleus of almost every cell in your body. • The structure of DNA.000 times as long as the cell itself.

high-tech procedures. • However. • Chromosomal DNA from a single cell is not visible to the naked eye. the amassed quantity can easily be seen and looks like strands of mucous-like. translucent cotton. when chromosomal DNA is extracted from multiple cells. .• Individual chromosomes can be studied using microscopes. but the double helix of a chromosome is so thin that it only be detected through innovative.

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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.• (Ethylenediamine tetracetic acid) is known as a chelating agent.

.• 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 come out of the suspension and may be seen and collected on a glass rod. .• DNA will be precipitated by adding cold alcohol to the cell extract.

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• Precipitate DNA. • Denature/digest proteins. • Separate contaminants (e. proteins. .. • Resuspend DNA in final buffer. Basic Steps in Isolating DNA from Clinical Specimens • Separate WBCs from RBCs. heme) from DNA. • Lyse WBCs.g.

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. heparin or citrate anticoagulant tubes to prevent clotting.5ml microcentrifuge tube.Procedure 1. Important: Blood must be collected in EDTA. transfer 300μl of blood to 900μl of Cell Lysis Solution in 1.

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Eppendorf tubes .

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Invert the tube 5–6 times to mix.. 2 3.000g for 20 seconds. 4. . Centrifuge at 13. Incubate the mixture for 10 minutes at room temperature (invert 2–3 times once during the incubation) to lyse the red blood cells.

Centrifuge .

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5. Approximately 10–20μl of residual liquid will remain in the 1.5ml tube. . Remove as much supernatant as possible without disturbing the visible white pellet.

Add 100μl of Protein Precipitation Solution to the nuclear lysate. . Add 300μl of Nuclei Lysis Solution. 7. Pipet the solution 5–6 times to lyse the white blood cells.000g for 3 minutes.6.Centrifuge at 13. and vortex vigorously for 10–20 seconds. 9. 8.Vortex the tube vigorously until the white blood cells are resuspended (10–15 seconds).

000g for 1. . 12 13. Decant the supernatant. . transfer the supernatant to a clean 1. 11. Centrifuge at 13.5ml microcentrifuge tube containing 300μl of isopropanol.minute and add 300μl of 70% ethanol to the DNA.10. Gently mix the solution by inversion until the white thread-like strands of DNA form a visible mass.

Centrifuge at 13. Gently invert the tube several times to wash the DNA pellet.14.000g for 1. 15 .minute 16. . Decant the ethanol and invert the tube on clean absorbent paper and air-dry the pellet for 10–15 minutes.

17 65°C for 1 hour or . Add 100μl of DNA Rehydration Solution to the tube and rehydrate the DNA. Incubating the solution at.overnight at 4°C Freeze at -20 or -70°C for long-term . storage .16.

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. Gel electrophoresis: •  Prepare 1% a garose gel and mix 10 l DNA samples with loading dye buffer as described in Lab 3. Loading DNA samples must be done slowly and smoothly to prevent sample from squirting up into the running buffer and diffusing away. Transfer the DNA samples into the wells of the gel using a pipette.

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