PortEco logo

Methods:Sonication

From EcoliWiki
Jump to: navigation, search

You can help EcoliWiki by editing the content of this page. For information about becoming a registered user and obtaining editing privileges, see Help:Accounts.

Contents

Purpose

Use high frequency sound wave to break down either cellular membranes and the release the contents of the cell or random fragmentation of DNA.


Share your knowledge and ideas. How you can help. See Help pages if you need help with using the wiki

Materials Needed

Sonicator

  • Bath Sonicator
  • Probe Sonicator

Sample (Protein or DNA)

Ice bath

Protocol

Warning.png

Ear protection should be worn when using sonication.

This includes everyone in the room while a sonicator is in use.

Beginning with an appropriate centrifuged culture:

Protein Extracts

  • If your protein is extracellular:
    • Keep the supernatant from centrifuged culture.
  • If your protein is intercellular:
    • Dispose of supernatant
    • resuspend pellet in an appropriate extraction buffer
      • Must preserve protein integrity and activity
    • Place sample container in water bath
      • Energy released into the cell suspension will be converted to heat. Therefore if the sample is not kept effectively cooled the sample and the container can be irreversibly damaged.
    • Place sample into sonicator, and set the intermittent pulses to the appropriate setting to break open the cultured cells.
      • If necessary add ice to the water bath to ensure proper cooling!
    • Spin down the sample - your protein should be in the supernatant.


DNA Fragmentation

  • Place sample in solution in appropriate contain (typically micro centrifuge tube)
    • To avoid nonrandom fragmentation at ends circulate and concatenated sample in by placing in ligation buffer.
  • Insert sonicator tip into solution as far as possible without touching the tube.
    • Keep sample in ice bath or the sample may be irreversibly damaged due to heat.
  • Set the amplitude as appropriate (higher amplitude will yield smaller fragments more quickly)
  • Use intermittent burst of sonication (example @ 5 μm: 5 seconds sonication, 15 seconds cooling[1])
    • Supercoiled DNA will take fewer bursts
  • Precipitate DNA
  • Fragment ends can be repaired with T4 DNA Polymerase
    • Do to the nature of this method the DNA does not have uniform ends for incorporation.

Links

Sample DNA Fragmentation Protocol

Textbook Explanation


User notes

References

See Help:References for how to manage references in EcoliWiki.

  1. Deininger, PL (1983) Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal. Biochem. 129 216-23 PubMed EcoliWiki page