5. After overnight growth, from plate B, we collect a single bacterial cell from

Question

5. After overnight growth, from plate B, we collect a single bacterial cell from a single colony and grow it in liquid culture for fast cell cycle and DNA multiplication. Then we extract DNA from cells and digest it with BamHI. We digest the DNA with BamHI and run the digestion products on a gel electrophoresis. What DNA band sizes would you expect to see?

A. 300, 750 bp

B. 750 bp

C. 2600 bp

D. 1050 bp

Read this for questions 1 through 6: In a gene cloning project, the goal is to produce a recombinant plasmid DNA that harbors the red segment of the genomic DNA specified in the following figure. The genomic DNA is 1600 bp, with multiple BamHI and EcoRI restriction sites. Digesting this DNA with BamHI enzyme results in multiple cut fragments of 300 bp, 750 bp, 75 bp, and other fragments of unknown sizes at both tails. The plasmid DNA, on the other hand, is 2600 bp long and contains only one BamHI restriction site on the Tetracyclin Resistant gene (TetRO, such that insertion of any forign DNA in this site results in "insertional inactivation" of TetR. In a test tube we digest the genomic DNA and plasmid DNA with restriction enzyme BamHI and then use the enzyme ligase with the hope to bring the red segment (750 bp) into the BamHI cut site of the plasmid DNA. Then, frozen E. coli will be heat shocked in the presence of this plasmid such that the bacteria is transformed with plasmid DNA which we hope contains the red-insert segment. We then plate the bacteria solution on to three types of plates A) without any antibiotic, B) with both ampicillin and tetracyclin antibiotic, and C with ampicillin only. 750 bp 300 b 75 bp 420 bp 730 bp 450 bp Bam HI EcoRI 2600 bp

Explanation / Answer

Answer: C. 2600 bp.

* The 2600 bp plasmid used in this given experiment having single restriction site for BamHI. During transformation experiment, recombinant DNA (2600 bp Plasmid + 750 bp insert) transformed successfully in host cell (E.coli) and resulting to produced both transformed cells with recombinant and non-recombinant transformed cells.

When these both cells are grown in a media containing Ampicillin and Tetracyclin antibiotic, only the non-recombinant transformed cells (resistant to both antibiotics) will grow in the plate. The recombinant transformed cells will not grow because TetR gene of recombinants was inactivated due to the insert of desired gene in the TetR region. If the non-recombinant DNA (2600 bp Plasmid without any insert) is then isolated from these non-recombinant transformed cells and digested again by BamHI, it will produce only 2600 bp (as the Recombinant DNA have single restriction site for BamHI).

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