4. At one time, plasmid pBR322 was used extensively in molecular cloning. Consid

Question

4. At one time, plasmid pBR322 was used extensively in molecular cloning. Consider the following sequence: a 1.2 kb fragment with PstI overhangs at each end was mixed with pBR322 that had been digested to completion with PstI and and DNA ligase was added. This mixture was used to transform E. coli cells. How would you select transformants that took up the desired plasmid? Be specific in your answer. (5 points). If you isolated plasmid DNA from several randomly-chosen colonies, what plasmid(s) would you expect to find and why? (5 points). After the initial selection, how could you further test the transformants for the presence or absence of the desired insert (apart from isolating plasmids and examining their structures by restriction digestion)? (5 points).

5. Indicate the final fate of each of the following cellular constituents during the plasmid isolation and purification method used in Experiment 5: a small, cytoplasmic molecule such as NADH; an integral membrane protein such as the lactose permease; a soluble, cytoplasmic protein such as triose phosphate isomerase; glutamine tRNA. For each species, briefly discuss the reason for its ending up in the fraction you suspect. (5 points).

Explanation / Answer

pBR322 is 4.36kb circular plasmid. if we inserted 1.2kb insert in it, the overall size of the plasmid will be

4.36+1.2 = 5.56kb.

now pBR has Amp and Tet resistant genes. digestion and insertion with Pst1 will destroy the Amp gene and insert containing plasmid will not be able to grow on Amp containing plate and we will get plasmid size of 5.56kb instead of 4.36kb.

we can test the transformants by restriction digestion. apart from this method replica plating, colony PCR and insert sequencing can also be performed.

during plasmid isolation, to release plasmid DNA we treat the cells with detergent and alkali so the cell membrane dissolve and the genomic DNA tangled and precipitated down with the cell debris and all other components.

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