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Grass genomic DNA extraction


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#1 meltvariant

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Posted 02 May 2013 - 03:31 PM

Hello, I am having a bit of trouble with a genomic DNA extraction and could use some advice in pinpointing the problem(s).

I have a larger number of grass (C4 tallgrass) samples that I intend to run through reduced-representation sequencing, on the order of 1000, with many more in the future. To handle this large number, I decided to go with Qiagen's 96 DNeasy plant kit. I found out too late however, that the best centrifuge available can induce up to 2250 *g, whereas the protocol recommends much greater force, 5800 *g. I decided to give it a trial run anyway, and doubled the spin times to try to compensate. The filter appeared to be dry after each of the spins. Unfortunately, however, the yield was far lower than hoped (~1.2ng/ml).

This could obviously be due to the centrifuge issue, or it could be that the bead beating was ineffective during lysis on Qiagen's TissueLyserII. While there was some clear evidence of cellular debris following the neutralization step, there wasn't as much as I am used to from doing bacterial mini-preps, and the leaves of grass still looked fairly intact.

Any thoughts on whether one, the other, both, or neither of these possible issues are the culprit?

Thanks in advance!

Edited by meltvariant, 02 May 2013 - 03:33 PM.


#2 mdfenko

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Posted 03 May 2013 - 03:50 AM

if the filter is drying then the problem is probably not the centrifugation (although, i would have at least tripled the time to make up for the lower g's).

check your lysis.
talent does what it can
genius does what it must
i do what i get paid to do




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