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General Genetics. Lab no. 6 DNA extraction from human blood. Objectives:. 1. Be introduced to the laboratory techniques involved in DNA extraction. 2. Test DNA integrity using gel electrophoresis. Introduction:.
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General Genetics Lab no. 6DNA extraction from human blood
Objectives: 1. Be introduced to the laboratory techniques involved in DNA extraction. 2. Test DNA integrity using gel electrophoresis.
Introduction: • 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 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.
Cont. • The more closely related organisms are, the more similar their DNA. Day to day, DNA’s 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.
Cont. • However, 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 structure of DNA, the double helix, is wrapped around proteins, folded back onto itself, and coiled into a compact chromosome.
Cont. • 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.
For extraction DNA some substances were used: • EDTA (Ethylenediaminetetracetic acid): • 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 and end product.
Cont. • Tris : • in the solution 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.
Cont. • SDS (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.
Cont. • NaCl (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.
Cont. • Alcohol: • 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.
Cont. • The DNA is visualised in the gel by addition of ethidium bromide. • This binds strongly to DNA by intercalating between the bases and is fluorescent meaning that it absorbs invisible UV light .
Materials: 1.Tric-CL 20 mM, Ph 7.6 2. Ethanol 70% 3. Isopropanol 4. Cell lysis buffer: • 10mMTris-Cl (Ph 8) • 1 mM EDTA (Ph 8) • 0.1% (w/v) SDS Store the buffer at room tempreture but chill an aliquot to 0◦C
Cont. 5. Potassium acetate solution: • 5 M potassium acetate 60 ml • Glacial acetic acid 11.5 ml • H2O 28.5 ml Store the buffer at room temperature.
Protocols 1.Transfer 300µl aliquots of whole blood to each of microfuge tube. 2.Add 900 µl of 20 mMTris-CL Ph 7.6 to each tube, Invert and mix. 3.Incubate the tubes at RT for 10 minutes, occasionally inverting the tubes. 4.Centrifuge at maximum speed for 20 sec at RT. 5.Discard all but 20 µl of each supernatant.
Cont. 6.Resuspend the pellets of white cells in the small amount of the supernatant left in each tube. 7.Combine the resuspended cell pellets in a single tube. 8.Add 600 µl of ice cold cell lysis buffer. Pippting several times. 9.Add 200 µl of potassium acetate solution and mix the contents by vortexing vigorously for 20 sec.
Cont. 10.Pellet the precipitated protein/SDS complex bycenrtifugation at maxium speed for 3 min at 4◦C . Apellet of protein should be visible at the bottom of the microfuge tube after centrifugation. If not, Incubate the lysate for 5 min on ice and repeat the centrifugation step. 11.Transfer the supernatant to a fresh microfuge tube containing 600 µl of isopropanol. Mix the the solution well and then recover the precipitate of DNA by by centrifuging the tube at maximum speed for 1 min at RT.
Cont. 12.Remove the supernatant by aspiration and add 600 µl of 70% ethanol to the DNA pellet .invert the tube several times and centrifuge the tube at maximum speed for 1 min at RT. 13.Carefully remove the supernatant by aspiration and allow the DNA pellet to dry in air for 15 min. 14.Redissolve the pellet of DNA in 100 µl of TE rehydration buffer. 15.The solubilization of the DNA pellet can be facilated by incubation for 16 hours at RT or for 1 hour at 65◦C.
