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Old 05-25-2012, 06:19 AM   #1
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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Default Covaris S2 cracking tubes.

Anyone had issues with a Covaris sonicator cracking tubes? We are using the microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm in a Covaris S2 that shipped with our SOLiD years ago.

No problems with it until a month ago when we noticed that the waterbath degasser was not functioning. Applied Biosystems FSE came and replaced the degasser pump motor. Seemed to be fine, except that about 1 in 3 of the micro tubes are cracking at the neck. Obviously not good. We have tried different lots of tubes -- same thing. So I presume it is an instrument issue.

Also, the 150 bp Covaris protocol seems to be shearing to ~75 bp.

Any ideas?

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Old 05-25-2012, 06:42 AM   #2
ETHANol
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I've sheared about 100 samples and had probably two tubes crack a little bit at the neck where the cap goes on. Nothing to mess up the experiment. I was shearing down to about 100 bp so I think this is about the limit of what the tubes can take. I imagine if you are shearing down to 75 bp, that is more then the tubes can handle. I don't think the amount of energy to shear smaller fragments is linear, so it seems like the machine is dishing out way too much energy.
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Old 05-25-2012, 06:53 AM   #3
pmiguel
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Yes, that seems to be what Covaris is thinking as well.

The only other explanation I can think of is that we do seem to have the most problems when the water bath has been degassing for hours. I always presumed that degassing hit a plateau beyond which there would not be much change in the "transparency" of the water to the acoustic energy. But maybe that is not the case? Maybe the instrument is calibrated to deliver the right amount of energy to the tube after 30 minutes of degassing, but after 4-5 hours it is delivering too much because the dissolved gas in the water has dropped much lower and transmits much more energy to the tube.

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Old 05-25-2012, 12:20 PM   #4
Hamid
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Hi Phillip,

There are several possible reasons for tubes breaking during processing on a Covaris instrument:
1. the Covaris microTUBEs are rated for use with a maximum duty cycle of 10% and intensity of 5. Using settings higher than the maximum will increase the probability of tubes breaking.
2. Incorrect water level. The proper water level when processing samples on the Covaris instruments using the microTUBEs is such that the water level is 1mm below the cap during processing. if the water level is lower, then the probability of tubes breaking will certainly increase.
3. The built-in intensifier in the holder is no longer straight. The purpose of the intensifier is to prevent the flow of directly waves from the bottom of the glass, and concentrate the waves from the surrounding area. If the intensifier is no longer straight ( perhaps due do damage caused by dropping the holder), then the probability of breaking the tubes will increase. This will also increase the energy reaching the samples, therefore reducing the shearing size range.
4. degass pump not working as efficiently. The purpose of the degass pump is to get rid of excess solubilized air from the water in the acrylic tank so that cavitation does not occur in the tank. If the degauss pump is not working efficiently, you will notice a shift to a higher size range for shearing. Since large cavitation bubbles do release very large amounts of energy, then that occurring on the outside of the tube on the edge of the tube and the glass might cause tubes breaking.

Thank you

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Old 05-27-2012, 11:02 AM   #5
pmiguel
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Thanks Ethan and Hamid,

It now appears that our older degassing pump was not doing a good job. As a result, during factory config of the unit, the "calibration factor" was set to a high value, 85%. This worked fine for years, until our pump died and was recently replaced by Applied Biosystems. This new pump, however, was much more effective. So effective that 85% was too high as a "calibration factor". We were hammering our glass micro tubes with too much energy and they were cracking at a high rate, as well as fragmenting our DNA shorter than expected.

Covaris gave us a new calibration factor, 75%, and instructions on how to input it into the unit EEPROM. Although we have not had a chance to test it extensively, none of the 4 test sample micro tubes cracked and the fragmentation appears to be back in the expected range for the standard protocols. So, looks like that was what happened.

My advice to anyone having their degassing pump replaced on one of these instruments is to do a few test samples and see that they are in the expected size range. If not you may have to contact Covaris and ask for instructions on how to adjust the calibration value.

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