Genetics Need details so I also understand 4. Your crazy cousin Bob has designed

Question

Genetics Need details so I also understand 4. Your crazy cousin Bob has designed a cloning project where he is inserting the C. elegans RICHES gene (named for a conserved amino acid domain) into a vector for expression in bacteria. Unfortunately, it's not working so well. You ask him to descrilbe each of his steps. a. Do a restriction digestion of the insert with the EcoRI enzyme b. Do a restriction digestion of the vector with the HindII enzyme c. Hybridize the two pieces using the DNA ligase enzyme What advice would you give Bob? Be sure to explain to him what he's doing wrong. why that would not work, and what you would suggest instead.

Explanation / Answer

The first problem is in the restriction enzymes because as he is using different enzymes to cut both vectors and dna, they do not produce the sticky ends to produce sticky ends. The cutting site is different for hind III and EcoRI. There are few enzymes that can produce sticky ends but EcoRI and hindIII are not one of them.

Second mistake is he is using an eukaryotic gene to be expressed in prokaryotelic system. Prokaryotes don't have introns in their gene and hence no system to remove introns from the eukaryotic gene and that is why the gene won't be expressed. For this matter he will need to use cDNA for the expression of genes into the bacterial system.

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