Alex, a Bio 311 student, decides she wants to clone DNA from her cat, Mr. Meowme

Question

Alex, a Bio 311 student, decides she wants to clone DNA from her cat, Mr. Meowmers, into a cloning vector. Alex PCR amplifies a segment of her cat's DNA and analyzes the sequence and finds that the cat DNA contains the restriction enzymes shown on the map below. Alex would like to clone in the largest possible fragment and she would like to use the same enzyme for both DNAs. Which enzyme should she use to cut both the vector and cat DNA? After Alex digests the vector and cat DNA, she mixes them together and ligates the fragments together with DNA ligase. She transforms the DNA into bacterial cells and plates the cells on ampicillin plates. Why does she plate the cells on amp plates? Next, Alex adds X-gal to her plates to determine which colonies are composed of cells that have taken up a recombinant plasmid. What color are the colonies that have taken up a recombinant plasmid? Would you expect a restriction enzyme with a longer restriction site to cut more or less often in the genome? More Often Less Often

Explanation / Answer

D.) A restriction enzyme with a longer restriction site is expected to cut LESS OFTEN. For example: a restriction enzyme with restriction sites of 6 bp , the cut or restrictions would occur at every 46 base pairs but an enzyme with a restriction of 8 bp will occur at an interval of every 48 bp, whose occurrence is less than that of the enzyme with 6bp restriction site.

A) The restriction site for the EcoRI is GAATTC, I.e. A 6 bp recognition site. For PstI is CTGCAG, 6bp,; For Sal1 is GTCGAC , 6bp and for Xba1 is TCTAGA

Alex should use Pst1 to cut both the vector and the cat DNA as the cat DNA contains the Pst1 endonuclease and the vector has a ampicillin resistance gene which is also present in the endonuclease.

B) After mixing the vector and the cat DNA together, she played them onto ampicillin played so as to check if the the vector has been incorporated into the cat DNA . If the vector is successfully incorporated into the cat DNA , the cells would succeed in growing on the ampicillin plates as they already have a gene for ampicillin resistance.

C) The recombinant plasmid would have a disrupted gene for lacZ as rendered by the vector. Plating them on a plate with Xgal would lead to the confirmation of the recombination as the recombinant DNA would give white coloured colonies. Since the restriction enzyme would cut the lacZ gene as it has an internal MULTIPLE CLONING SITE, the lacZ gene would be dirupted and the foreign DNA would be inserted within the lacZ gene. Thus no beta galactosidase will be formed and there would be no production of blue colour.

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