View Single Post
Old 03-05-2015, 03:47 PM   #18
kerplunk412
Senior Member
 
Location: Bioo Scientific, Austin, TX, USA

Join Date: Jun 2012
Posts: 119
Default

Quote:
Originally Posted by nucacidhunter View Post
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.
Your logic makes sense to me, so maybe the difference in size before and after vortexing does not explain my observations. However, I tested this fairly rigorously and a few of my colleagues have tried this as well, so I can say with confidence that with the gDNA samples I was working with a gentle flick was not enough to get a consistent reading, vortexing was required. As far as damaging the DNA, I am pretty sure 10 seconds of vortexing will not cause enough DNA fragmentation to matter for most NGS applications. If it was that easy to fragment DNA into small pieces no one would need to buy a Covaris!

Edit: I should also mention that the variation seen before vortexing was at most about 2x. Variation after vortexing was ~1%.

Last edited by kerplunk412; 03-05-2015 at 03:52 PM.
kerplunk412 is offline   Reply With Quote