Question

After applying the selection process, your bacterial cells may contain at least three different types of common possible recombinants.

a. What restriction enzyme(s) could you use to distinguish between the various recombinants?
b. Draw and label a rough sketch of an agarose gel pattern that you would see if you performed the restriction digests of possible AmpR recombinants. Also provide plasmid maps to show how you calculated the sizes of fragments. Be sure to draw a DNA ladder and label fragments with their estimated size.

Answer 1

1. Restriction enzymes are endonucleases, that is, enzymes that digest nucleic acids. Restriction enzymes recognize specific sequences of nucleotides in a DNA strand. Their use allows the detection of point mutations in DNA and eliminates the need for subcloning and sequencing.

Traditionally, four types of restriction enzymes are recognized, designated I, II, III, and IV, which differ primarily in structure, cleavage site, specificity, and cofactors.

b. answer is attached.

c. Multiplying the genome size by the frequency equals the number of restriction fragments produced, or (2.5 x 107 bp)(2.56 x 10-4 bp-1) = 6400 fragments. Divide the genome size by the number of fragments to determine the average fragment size, or 2.5 x 107 bp/6400 = 3.9 x 103 bp.]