The preliminary steps
Before you begin, here is a quick refresher on how you get to the starting point of this activity:
- Enzyme E sits within the MCS (Multiple Cloning Site - an artificial DNA sequence within a vector that is engineered to contain a range of restriction enzyme recognition sequences ("enzyme sites") - this facilitates the cloning of insert DNA). You can see the MCS below in the 'Plasmid map prior to cloning of insert' diagram.
- The insert was initially cloned by PCR using primers engineered to contain enzyme E sites.
- The vector and insert were digested with enzyme E, mixed together and ligated to produce the final plasmid construct which you'll be working with during this activity.
- On this map you can see position numbers for the various enzyme sites. The bracketed numbers show the enzyme sites for the vector (black) and insert (green) prior to cloning the insert. Once the insert is cloned into the vector, the relative position numbers of the enzymes sites changes. This has been calculated for you - the bold red numbers represent the enzymes positions on the final plasmid construct (i.e. vector+insert). For example, notice how enzyme site B started as position (1997) in the vector alone, but after the insert is added it changes to 4051 - this makes sense because the insert is 2054bp (and 1997 + 2054 = 4051!). Understanding this concept is important for predicting fragment sizes after restriction enzyme digestion.
Select the Vector, Insert, MCS sections and the Plasmid label on the diagrams below and read the explanations provided. Once you've done this you should then begin the first part of the activity.