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. ...Show more