Table of Contents

• Prequiz
• 1: Three thieves and a beaded necklace
• 2: And the moral is?
• 3: A real plasmid
• 4: Practice with restriction sites
• 5: What's wrong with these gels?
• 6: Playing with plasmids
• 7: Other weird things
• 8: Summary

... and the moral is?

OK, so here are some parallels:

Beaded Necklace: Like a beaded necklace, each plasmid is a circular piece of DNA, made up of four bases in a non-predictable pattern.

The Thieves: The thieves chopped up the necklace; likewise, restriction enzymes chop, or cut, a plasmid.

Two Blue Beads: The thieves chopped every time they saw a certain pattern of beads; likewise, restriction enzymes recognize certain sequences of bases, called a restriction site, although these sequences are generally four to eight bases in length, not just two.

The Length of the Pieces: One way to learn about the plasmid and its fragments is to find out how long those fragments are. Although scientists are smarter than thieves, DNA itself is too small to see – so you can’t simply count the number of bases in a fragment, which leads to …

The snail race: in order to determine the length of the fragments, scientists use gel electrophoresis. Bottom line in electrophoresis: small molecules move faster, big molecules move slower, so by seeing where the fragments end up after a short period of time, you can guess how big they are. The approximate size of each fragment can be determined by comparison with a set of standards of known size (DNA ladder) run on the same gel.

 

Before we go on to the plasmid, check your understanding:

• A connected circle with five recognition sites
  would get broken into pieces
• A linear strand with five recognition sites
  would get broken into pieces
• If a connected circle with three red recognition sites and five blue recognition sites is broken at the red sites ONLY,
  how many fragments will result?
• If the same connected circle is broken
  at both red and blue sites?