A plasmid DNA and a linear DNA (both are of the same size) have one site for a restriction endonuclease. When cut and separated on agarose gel electrophoresis, plasmid shows one DNA b and, while linear DNA shows two fragments. Explain.
When a plasmid DNA and a linear DNA having one site for a restriction endonuclease are cut and separated, plasmid shows one DNA band, while linear DNA shows two band because of difference in their basic structure.
Plasmid is a circular DNA molecule and when cut with these enzyme, it becomes linear but does not get fragmented due to presence of only one restriction site, whereas a linear DNA molecule gets cut into two fragment.
How does one visualise DNA on an agarose gel?
A compound called ethidium bromide stains DNA, which on exposure with ultra-violet, (uv) radiation gives orange light band of DNA. Hence, DNA fragments appear as orange band in the presence of ethidium bromide and UV light.
A plasmid without a selectable marker was choosen as vector for cloning a gene. How does this affect the experiment?
In a gene cloning experiment, first a recombinant DNA molecule is constructed, where the gene of interest is ligated to the vector (the step would not be affected) and introduced inside the host cell (transformation).
Since, not all the cells get transformed with the recombinant/plasmid DNA, in the absence of selectable marker, it will be difficult to distinguish between transformants and non-transformant, because role of selectable marker is in the selection of transformants.
A mixture of fragmented DNA was electrophoresed in an agarose gel. After staining the gel with ethidium bromide, no DNA bands were observed. What could be the reason?
The reasons are as follows
(i) DNA sample that was loaded on the gel may have got contaminated with nuclease (exo or endo both) and completely degraded.
(ii) Electrodes were put in opposite orientation in the gel assembly that is anode towards the wells (where DNA sample is loaded). Since, DNA molecules are negatively charged, they move towards anode and hence, move out of the gel instead of moving into the matrix of gel.
(iii) Ethidium bromide was not added at all or was not added in sufficient concentration and so DNA was not visible.
Describe the role of $\mathrm{CaCl}_2$ in the preparation of competent cells?
$\mathrm{CaCl}_2$ is known to increase the efficiency of DNA uptake to produce transformed bacterial cells. The divalent $\mathrm{Ca}^{+2}$ ions create transient pores on the bacterial cell wall by which the entry of foreign DNA is facilitated into the bacterial cells.