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Old 04-27-2011, 07:22 PM   #1
seqgirl123
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Default shearing question using Covaris

Would like to understand something when I use Covaris for shearing.

I have DNA that has been diluted to 5ug in 120ul for shearing or 41ng/ul. I shear and measure concentration by Picogreen. But it is about 30ng/ul after shearing. Why does the concentration drop? It is only sheared DNA, not cleaned. Is it due to the DNA being less compact after shearing? Does it have anything to do that DNA is fragmented now and this causes the concentration to drop? I have tried other DNAs and their concentrations drop just like this.
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Old 04-27-2011, 08:11 PM   #2
ECO
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Single stranded regions. (?)
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Old 04-28-2011, 05:51 AM   #3
LMcSeq
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Agreed-PicoGreen is intended to measure double stranded DNA. If the shearing resulted in any single stranded material, that would not be detected.
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Old 04-28-2011, 11:17 AM   #4
Hamid
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Hi seqgirl123,

What you are noticing is how pico green method is used for concentration determination. Two points of interest:

1. Pico green binding to DNA and its intensity is dependent on the size of DNA. It is recommended that you generate a standard curve using DNA in in the same size range as the distribution of your fragments after shearing. If you use a standard curve that has been generated using large DNA such as lambda, the concentration from that standard curve will not apply accurately to the smaller sheared material.
2. Quite a few service labs use a combination of pico green and A260/A280 to check the quality and concentration of their samples. If you test the samples you mentioned on a nano drop or equivalent system, you will notice that there is very very minimal loss of your samples.

Thank you

Hamid
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