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Grass DNA extraction issue

Grass DNA extraction issue - DNA Techniques

Grass DNA extraction issue - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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Old 05-02-2013, 11:20 PM
Pipette Filler
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Default Grass DNA extraction issue



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?

One additional note: these leaves were dried and stored using silica gel beads last summer, so they are bit aged, but not unheard of.

Thanks in advance!
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