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Old 03-05-2015, 12:58 AM   #16
Jafar Jabbari
Location: Melbourne

Join Date: Jan 2013
Posts: 1,234

Originally Posted by kerplunk412 View Post
I have noticed something similar when quantifying gDNA by Nanodrop. What I saw was values that would vary quite a bit when reading different "drops" from the same tube of gDNA. This was solved by vortexing the DNA for 10 seconds. The idea is that the genomic DNA molecules are so large that one microliter might have varying amount of these large DNA molecules. Following vortexing the gDNA is in much smaller fragments, which allows it to exist more evenly in solution such that every microliter will have a much more similar amount of DNA. Think of it of grabbing handfuls of sand versus handfuls of medium sized rocks and weighing them. The handful of sand will be very close to the same weight each time, but the rocks will vary much more. I haven't tested the theory about large DNA vs sheared DNA, but we have tested vortexing DNA for 10 seconds prior to reading on the Nanodrop and it definitely results in much more consistent readings.
Mass of genome in one human cell is 6.6 pg, so in a DNA solution of 10 ng/ul we would have equivalent of DNA from 1515 cells which would be 45,450,000 fragments of 100kb. Most standard extraction methods will result in fragments less than 100kb. So, I do not see how one can justify that 45.5 million fragments in 1ul will aggregate in a solution to give 10x variation in consecutive reads. I think just a gentle flick would be enough to have a homogenous solution (if sample was frozen) and vortexing definitely would damage large DNA fragments.
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