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Old 04-18-2012, 10:07 AM   #5
Hamid
Senior Member
 
Location: San Diego, CA

Join Date: Sep 2009
Posts: 105
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Dear Gajz,

I apologize for the delay in responding to your post.
While seemingly beneficial to use a low cost PCR tube in your Covaris instrument, it is critical to address the energy requirement to generate small DNA fragments. Covaris specifically engineered the microTUBE to work in conjunction with AFA to control the energy for generation of small DNA fragments:

1. The amount of energy required to shear DNA is inversely proportional to the size of the DNA fragments desired. For example, large fragments such as 3kb require low energy and consequently, plastic may be used (e.g., Covaris miniTUBE). More importantly, however, is that to generate small fragments of DNA, a high level of energy is required. If this energy is not efficiently transferred to the sample and if the subsequent heat is not readily dissipated the sample will quickly heat. As a result, for shearing to NGS-size fragments it is essential to maintain an isothermal processing temperature during shearing by controlling the thermal events through BOTH hardware (Covaris AFA) and the consumable (microTUBE). These tubes are specifically designed to work with the acoustic circuitry to maintain an isothermal environment during DNA shearing

2. Plastic materials such as polypropylene PCR tubes absorb acoustic energy and then through convective heat-transfer your sample is heated. Your sample may be in an iced water bath, but your internal sample fluid is heated. The heat is then prevented from efficiently dissipating from the sample since the polypropylene then acts as an insulator. I have attached a slide of the temperature mapping of DNA samples sheared to 200pb in the Covaris microTUBEs compared to the same sample processed in a think wall 200ul PCR tube. As you can see, even though the PCR tube sample is in a 6C water bath, the temperature of the sample is greater than 50C. In comparison the temperature of the sample in the microTUBE using the same settings is the same at that of the surrounding water bath.

3. The lack of the thermal control when using the PCR tubes/plates will certainly effect reproducibility, shearing size distribution, total DNA recovery, and will cause thermal biased shearing of samples in regions with low GC content.

4. Several large sequencing centers did initially evaluate using PCR plates instead of the microTUBE plate, but observed shearing inconsistency, and failed sequencing runs which they attributed to the shearing in the PCR plates.

The microTUBE is an integral component of the AFA circuit.

Thank you

Hamid
Attached Files
File Type: pdf microTUBE vs PCR tube with AFA.pdf (175.9 KB, 84 views)

Last edited by Hamid; 04-18-2012 at 10:24 AM.
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