Running Restriction Digests on the Phage Genomes

Objective:

To broadly characterize the genomes of isolated phages.

Rationale:

Each phage genome is unique. While we will be sending the phages for sequencing for in depth analysis, restriction digests provide another way to view differences between phage genomes. In this assay we will take advantage of enzymes from bacterial phage defense systems, known as restriction endonucleases. These restriction endonucleases cut specific sequences of DNA often found within phage genomes. Phages can have one or more of these sites scattered throughout their genomes, which means that when we cut with these enzymes, the resulting pieces will be of different sizes. We can therefore take the cut DNA from a given phage, and separate it by size, and see how many pieces there are and how big they are, making up a kind of fingerprint for each phage. By comparing different digested genomes we can then determine whether the phages we are working with have the same or similar “fingerprints”. This helps us broadly determine if we have different phages as we expect that only very closely related (nearly identical) phages would have the exact same fingerprints.

Before you start:

  • You need to have purified phage DNA
  • You need to know the concentration of your phage DNA (use nanodrop)
  • You need to have thawed enzyme buffer

Materials for Restriction digests

  • Phage DNA
  • Sterile water for diluting phage DNA
  • Microcentrifuge tubes
  • Cutsmart Buffer
  • BamHI Restriction Endonuclease
  • HindIII Restriction Endonuclease

Procedure

  1. Determine the volume of DNA you need to add to your reaction to get 200ng. We only want to add 1,000ng to ensure that we can easily compare samples later and so that we don’t add more DNA than the enzymes can cut.

    The equation you need to use is \(volume = \frac{1,000} {concentration}\)

  2. For each sample:
    • If the calculated volume was less than 17µL: add the volume of phage DNA equal to the calculated in step 1. Add enough sterile water to bring the volume in the tube up to exactly 17µL. For example, if you needed 19µL to get 1,000ng of DNA, then add 21-19=2µL of water to your tube. In the end each tube should have 17µL after this step.
    • If the calculated volume was more than 17µL: Simply add 17µL
  3. Add 2µL Cutsmart buffer to each tube
  4. Add 0.5µL HindIII to each tube
  5. Add 0.5µL BamHI to each tube
  6. Briefly vortex to mix, then lightly spin on the microcentrifuge
  7. Place in heat block at at 37°C for 30minutes
  8. Freeze at -20℃ until we are ready to analyze.