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DNA is present in the cells of all living organisms. This procedure is designed to extract DNA from onion in sufficient quantity to be seen and spooled. It is based on the use of household equipment and supplies. DNA is extracted from human cells for a variety of reasons. With a pure sample of DNA you can test a newborn for a genetic disease, analyze forensic evidence, or study a gene involved in cancer. Try this virtual laboratory to perform a cheek swab and extract DNA from human cells.DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and one optional steps in a DNA extraction:
Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by grinding or sonicating the sample.
Removing membrane lipids by adding a detergent.
Removing proteins by adding a protease (optional but almost always done).
Precipitating the DNA with an alcohol - usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.
Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg2+ and Ca2+, which prevents enzymes like DNAse from degrading the DNA. Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation. If desired, the DNA can be resolubilized in a slightly alkaline buffer or in ultra-pure water.
For onion, the concentration that obtained is as follow:
Concentration = 47.8 ng/Âµl
A260 = 0.975
A280 = 0.717
A260/280 = 1.33
A260/230 = 0.69
Since A260 reading of 1.0 = 50 ng/Âµl dsDNA
Thus, A260 reading of 0.975 = 47.8 ng/Âµl
[DNA] diluted sample = 47.8 ng/Âµl dsDNA
[DNA] original sample = 6 dilution factor x 47.8 ng/Âµl
=286.8 ng/Âµl dsDNA
Total amount of DNA extracted formed = 286.8 ng/Âµl x 6000 Âµl
= 1720800 ng DNA
Bead beater is used in the breaking apart or "lysing" of cells in the early steps of extraction in order to make the DNA accessible. Glass beads are added to an eppendorph tube containing a sample of interest and the bead beater vigorously vibrates the solution causing the glass beads to physically break apart the cells. Other methods used for lysing cells include a french press and a sonication device.
A centrifuge such as this can spin at up to 15,000 rpm to facilitate separation of the different phases of the extraction. It is also used to precipitate the DNA after the salts are washed away with ethanol and or isopropanol.
A gel box is used to separate DNA in an agarose gel with an electrical charge. When the red and black leads are plugged into a power supply the DNA migrates through the gel toward the positive charge due to the net negative charge of the molecule. Different size pieces of DNA move at different rates. The larger pieces moving more slowly through the porous medium, thereby creating a size separation that can be differentiated in a gel.
The DNA double helix cannot be see by naked eye because it is too small to be seen with the naked eye. It can be extracted as millions of strands of DNA. DNA is come from 99% nucleus of the cell. The DNA can be store by extract their DNA with a wooden skewer and place it in a small, sealable container holding alcohol. As long as the container is tightly sealed and is not shaken much, the DNA should remain. However, there is a chance that the DNA could continue to degrade if any enzyme is transferred with the DNA into the new container.
It is difficult to dissolve DNA because DNA has the structure of double helix, with about 10 nucleotide pairs per helical turn. Each spiral strand, composed of a sugar phosphate backbone and attached bases, is connected to a complementary strand by hydrogen bonding (non- covalent) which has a very strong bond between paired bases, adenine (A) with thymine (T) and guanine (G) with cytosine (C). Adenine and thymine are connected by two hydrogen bonds (non-covalent) while guanine and cytosine are connected by three. In order to dissolve the DNA, an organic compound such as alcohol and chloroform can be used. Besides that, enzyme such as Proteinase K also can be used to increase the speed of dissolving the DNA.
Structure of DNA
DNA will become even more difficult to dissolve if it is allowed to dry for a long a period. It can be improve by dissolve it when the DNA is wet. The long after isolation or alcohol wash also may be useful. DNA that is dissolve in TE at 56Â°C can also make the work easier
A new method CTAB-silica for DNA extraction and purification from the leaves and buds of Castanea mollissima and Ginkgo biloba was tested. The method is based on the silica-based purification protocol developed by Boom et al. (1990). By modifying the protocol, plant genome DNA could be extracted easily from dormant buds, mature leaves, and other parts of plant. The results showed that the purified DNA was of high purity and could be analyzed by PCR. Furthermore, this CTAB-silica method took much less time for a successful DNA purification process compared to the traditional methods (CTAB and SDS). By the method, the suitable DNA can be extracted and purified from over 10 plant samples by one person in an hour.
Besides that, the proteins also must remove by phenol extraction, followed by chloroform extraction, before ethanol precipitation. Otherwise, the protein-DNA complex is hard to be re-dissolved and the pellet also can dissolve in a warm water (37C) bath than in the refrigerator. Proteinase K also can used to treat the DNA pellet after extraction. Then re-extract over chloroform and re-precipitate
The types of reagent used and its purpose in the DNA extraction is summarize as follow:
Salt provides the DNA with a favorable environment; it contributes positively
charge atoms that neutralize the normal negative charge of DNA. The salt causes the
precipitation of proteins and carbohydrates located in the produce, and help to separate
The cell membrane is composed of lipids (fats) and proteins. The detergent acts on
these lipids like it does with other fats (think of how detergent works with grease on
dirty dishes) and captures the lipids and proteins. In the process of capturing these
molecules, the cell membrane is lysed (or broken), destroying the membrane and
release the parts of the cell into the solution.
Help to break down the cell walls
DNA will not dissolve in this alcohol, so the DNA comes out of the solution, or
precipitates. It is less dense than water or cell scum--which is what settles to the bottom
of the glass--so it floats up into the alcohol layer, where you see it as a snotty,
string-like substance, with small bubbles formed on it.
Protein is stored in it for the nutrition of the new plant
Acts as an enzyme when interacting with the solution. DNA, which is usually contained
in the cell as long strands wrapped around proteins, needs to be unwound in order to be
completely separated from the proteins and other cell contents. The meat tenderizer acts
like an enzyme in that it cuts out the proteins from the DNA-protein complex.
NaCl provides Na+ ions that will block negative charge from phosphates on DNA.
Negatively charged phosphates on DNA cause molecules to repel each other. The Na+ ions will form an ionic bond with the negatively charged phosphates on the DNA, neutralizing the negative charges and allowing the DNA molecules to come together.
EDTA is a chelating agent and has great affinity with matel ions and Mg-ion present in DNase as a cofactor and responsible for DNase degradation action of the DNA, then EDTA will bind with Mg-ion and complete the action of DNase.
Phenol/chloroform extraction is an easy way to remove proteins from your nucleic acid samples and can be carried out in a manner that is very close to quantitative. Nucleic acids remain in the aqueous phase and proteins separate into the organic phase or lie at the phase interface.
In general, DNA can be isolate by the method of large scale double-stranded DNA isolation, midiprep double-stranded DNA isolation, miniprep double-stranded DNA isolation, large scale M13RF isolation, single-stranded M13 DNA isolation using phenol, Biomek-automated modified-Eperon isolation procedure for single-stranded M13 DNA, 96 well double-stranded template isolation and genomic DNA isolation from blood. Whereas the basis purification methods is using silica, which is called the CTAB-silica for DNA extraction and purification.