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.
What would happen when one grows a recombinant bacterium in the bioreactor but forget to add antibiotic to the medium in which the recombinant is growing?
In the absence of antibiotic, there will be no pressure on recombinants to retain the plasmid (containing the gene of our interest). Since, maintaining a high copy number of plasmids is a metabolic burden to the microbial cells, it will thus tend to loose the plasmid.
$$ \text { Identify and explain } A, B \text { and } \mathrm{C} \text { in the PCR diagram given below. } $$
$$ \text { Region to be amplified } $$
In PCR, each cycle has three steps
(i) Denaturation of DNA Sample Unwinding of two strand of DNA by heating the sample at $92-94^{\circ} \mathrm{C}$.
(ii) Primer Annealing Primers get positioned on the exposed nucleotides as per base pairing rules.
(iii) Extension of Primers DNA polymerase recognises primers as 'start' tags and begins to extend the primers using the free nucleotides provided in the reaction and the genomic DNA as template.
With each round of reactions, the DNA doubles.