Lidstrom:Sonicator

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Basics

• sample should be on ice during sonication and between sonication rounds to preserve the activities of the proteins you are extracting.

How should I do the pulses?

• The design criteria for the pulsing are: (a) enough seconds of sonication to actually break open cells (b) keeping the samples as cool as possible while this happens.
  • Ceci advised that keeping the temperature below 10oC is good enough.
  • In principle for a given sample size there are frequencies at which you can pulse and wait that will not lead to net increase in temperature (read after sonication) over time.
• You can measure the temperature of the samples with a heat-block style stick/alcohol thermometer
  • If you want to know whether your sample is above or below 10^oC, it is best to start with the thermometer close to 10oC so you can see whether the reading goes up or down after insertion into the sample. If your thermometer is too hot or too cold than the time it takes for the sample to reach equilibration with the thermometer has allowed for a lot of time for the sample to be cooled by the ice it should be sitting in. Also, if the thermometer is too hot, you are adding heat to your sample unnecessarily.
• Janet had programmed the sonicator to pulse for 1 sec, and not pulse for 30 seconds and that was sufficient to not heat up samples w/ volume = 1/2 mL.
  • On for one sec and off for <10 sec definitely leads to excessive heating.

How many pulse seconds should be applied per sample?

• This is actually a tricky question that requires better data than anyone in our lab seems to have. The best test would be to have many tubes of biomass, test different amount of pulse seconds, then measure the protein concentration with BCA to see if there is an asymptotic relationship between the total number of seconds and the resulting soluble protein concentration.

What volume of cells should I use?

• ~1/2 a mL is suitable for our sonicator tip size.

How deep should the shaft be?

• You don't want it to foam, so it should be near but not touching the bottom of the eppendorf tube.