Plasmid Isolation and Restriction Digest

Plasmid Isolation and Restriction Digest

General questions on plasmids:

  1. In your own words, briefly explain what a plasmid is and why they are important in bacterial genetics. (2 pt)


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  1. Plasmids cost the bacteria energy to replicate and maintain, sometimes causing the bacteria to reject the plasmid. How do we ensure the bacteria retain the plasmids? (2 pt)


Questions involving BLAST search

After isolating the plasmid, your TAs have sent it off for sequencing. The sequence has been posted under the Activity 3 module as “plasmid_seq.txt”. Copy-paste this sequence into a nucleotide BLAST search and answer these questions based on the first result.

  1. What is the query cover of our result? What fraction of nucleotides match between our query sequence (the sequence you put into the search) and the database sequence found in the search? (1pt)


  1. Click the “GenBank” link to find more information on the database sequence. What is the title of the first reference relating to this sequence? (1pt)


  1. The “Features” section of a GenBank page lists the genes found in the returned sequence. Give the abbreviation of each gene, , and a short description of what the gene does/codes for (google may help with the last part). (3pts)


Questions for after week 2 (Restriction Digest)

  1. List the restriction enzymes that were used in this lab, their target sequences and the location(s) on the plasmid of those target sequences. (1 pt)


  1. Other than visualization on a gel, describe a reason for cutting a plasmid at a single point. (1 pt)


  1. Other than visualization on a gel, describe a reason for cutting a plasmid on either side of a gene (1 pt)

Use the sample ladder diagram below as reference for the ladder on your gel when estimating DNA sizes in the following questions

  1. Despite being the same number of base pairs, a circular plasmid and a linearized plasmid will move different distances down the gel. Compared to the linearized plasmid, does the circular plasmid appear as though it is larger or smaller? What form is the circular plasmid in and why do they move differently despite being the same length? (2 pt)


  1. For one of the restriction digests, you used the BamHI enzyme. How many bands do you see? What are their approximate sizes? (1 pt.)