Deciphering Diagnostic Digests

This activity has been designed to prepare you for the lab component of Practical C. It will help you understand the concept of carrying out diagnostic digests using restriction enzymes.

By completing this activity you will be able to correctly predict the expected number and size of DNA bands that will appear on an agarose gel for a given combination of restriction enzyme(s), vector(s) and insert DNA sequence(s). When correctly applied, this understanding will allow you to determine the orientation (i.e. “forward” or “reverse”) of the insert DNA within a supplied plasmid construct.

This task should take between 10 and 20 minutes to complete. Here's what you'll be doing during this task:

  1. First you will review the terms Vector, Insert, Plasmid and Multiple Cloning Site (MCS) as these are central to your understanding of the experiment to be carried out in the laboratory.
  2. You'll then do a quick refresher on the preliminary steps required to produce the final plasmid construct you will be working with during this activity. You'll also be able to familiarise yourself with the terms to understand where they're located and what they do.
  3. You'll be able to analyise some supplied "Insert DNA" from a tet file and determine the enzymes that digest this DNA.
  4. You'll then be asked to select two different enzymes that will help you to determine the orientation of the insert. You'll be supplied with all the information you need to calculate the digest fragment sizes for enzymes used alone ("single digest") or in combination ("double digest).
  5. Once you've correctly calculated the single and double digest fragment sizes for the two chosen enzymes, you'll then be asked to predict the gel electrophoresis results by marking the expected band locations on a gel.
  6. Finally, you'll be presented with a series of gel electrophoresis results and will need to determine the enzymes that were used to generate the observed restriction patterns.

Throughout this task you'll be guided through each step and have the chance to check your calculations and selections as you go.

Select the 'Start' button when you're ready to begin.

Start