McClean: Sonication Yeast

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Overview

Protocol for sonicating yeast for microscopy (and other applications) where you want the sonication to maximally break up clumps while maintaining 100% viability.

Our sonicator is a Fisher Scientific Model 705 Sonic Dismembrator.

Materials

  • Mid-log (or other stage of growth) cell culture
  • 1.5 ml eppendorfs
  • Sonicator
  • Hearing protection


Protocol

  1. Clean the sonicator microtip by wiping it down with 70% ethanol and a kimwipe
  2. Aliquot ~500 μL cells into 1.5 ml eppendorf tube. DO NOT USE A GLASS TUBE
  3. Submerge the sonicator microtip into the eppendorf.
    • Aim to have the tip ~ 1 cm from the bottom of the tube. Too deep and you won't get adequate mixing. Too shallow and you run the risk of foaming
    • Center the tip inside the eppendorf
    • MAKE SURE THE TIP IS NOT TOUCHING THE WALLS OR BOTTOM OF THE TUBE
  4. Turn on the sonicator
  5. Press "Yes" that you are using a microtip
  6. Select to modify a program or sequence
  7. Select/Modify a program
  8. Program 1 has the Maitreya lab protocol saved
    • Amplitude: 5
    • Pulse-ON Time: 1 sec
    • Pulse-OFF Time: 1 sec
    • Elapsed Time: 20 sec
  9. WEAR EAR PROTECTION
  10. Press Start
  11. Monitor the tube to make sure there is no foaming. If foaming does occur:
    1. Stop the sonicator
    2. Centrifuge the tube until the foam has dissipated
    3. Vortex to resuspend the cells
  12. After sonication, clean the microtip with 70% ethanol and kimwipe



Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

*Megan N McClean 18:52, 8 March 2017 (EST) Two places to start in determining a good protocol for our QSonica sonication protocol are below. The Botstein Protocol is the one that I used previously at Princeton. Maitreya's seems very similar except for longer pulses and longer rests.

Botstein Lab Protocol: Sonicator unknown, Program #1, 5 sec pulse, pulse on 0.5sec, off 0.5 sec, output level 5

Maitreya Lab protocol: Misonix S4000, Program #1, which consists of 10, 1 second bursts at Amplitude=5, with a 1 second rest in between bursts. This seems to separate cells nicely

  • Taylor D. Scott 16:56, 20 March 2017 (EDT): I tested the Maitreya lab protocol (amplitude = 5, 1 sec on, 1 sec off, 10 periods) on a Sigma strain and it separated the cells nicely. I had 4% dead cells after sonication.

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