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.
Use high frequency sound wave to break down either cellular membranes and the release the contents of the cell or random fragmentation of DNA.
- Bath Sonicator
- Probe Sonicator
Sample (Protein or DNA)
Beginning with an appropriate centrifuged culture:
- 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.
- 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)
- 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.
See Help:References for how to manage references in EcoliWiki.