One purine pair with one Pyrimidine with hydrogen bond to make the double stranded DNA. Adenine (A) pairs with Thymine (T) with double H-bond and Guanine (G) pairs with Cytosine (C) through triple H-bond.
Isolation procedure requires disruption of cells so that the cell content comes out, followed by sedimentation of the cellular debris on application of centrifugal force and to collect the DNA from the supernatant. These DNA fragments are separated using gel electrophoresis. The process encompasses separation, based on their size, the pore size of the gel, the voltage gradient applied and the salt concentration of the buffer. Larger pore size is for the separation of fragments larger than 500- 1000 bp and smaller pore of agarose gels are used to resolve fragments smaller than 1000 bps and can be visualized. The technique of electrophoresis is based on the fact that since DNA contains phosphate group, it is negatively charged at the neutral pH. When electric potential is applied, it moves towards the positive terminal.
The solidified agarose gel is inserted into the electrophoresis chamber and is just covered with buffer. The DNA sample is mixed with the loading buffer and then pipette in the sample wells. On application of the current DNA migrates towards positive (red colored) electrode. The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes. After adequate migration, DNA fragments are visualized by ethidium bromide. This is a fluorescent dye and it intercalates between the bases of DNA and RNA. It is incorporated in the gel so that staining occurs during electrophoresis. Bands appear on the gel and can be visualized.
A fluffy white layer was formed at the boundary between the green and the purple liquids when the ethanol was added. It was made up of fine filaments.
After putting the electrical current, strands of the DNA become visible to the naked eye. It becomes like stains, or bands, on the gel.
NaCl removes protein and carbohydrate in DNA and also act as lysing buffer. NaCl contains Na+ which binds with the negatively charged phosphate molecule of the DNA. It also stabilizes the pH and process the density of DNA. Washing liquid reduces the acidity of solution and remove CO2.
Detergents remove the interfering cells and are used as a substitute for the chemical compound that is capable of damaging the cell wall and membrane. They act as emulsifying agents and can digest compound that causes stiffness of polymeric cells.
Endiamin tetra ethyl acetate (EDTA) serves to remove the Mg+2 ion and proven enzymes which can damage cellular DNA, it protects the DNA from DNAse. It interrupts the interaction of polar cell membrane and unites as detergent.
Gel electrophoresis is a powerful tool for the separation of macromolecules with different sizes and charges. DNA molecules have an essentially constant charge per unit mass thus they separate in agarose, based on the size, smaller the size more distance it can travel and larger the size of the DNA less it can travel. Increasing the concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. The