Hi all,

Recently, I have performed DNA extractions of some viola and fern samples using the Qiagen DNeasy Plant Mini kit. Unfortunately, A260/230 nm ratios are poor (< 1.4) for all viola samples and for some of the ferns. Attached a spectrum showing a shift of the peak to the left (~ 250 nm), while the trough is still at 230 nm. I have quite a hard time figuring out the cause of this shift, as it does not fit the shifts described in the literature. I don’t think that polyphenols are causing this issue as they would result in the maximum being shifted to the right. In addition, carry-over of guanidine (present in Buffer AW1) is unlikely to be a problem, as I already perform three washing steps with Buffer AW2 with 5 min incubation each.

Here some key points of the protocol used:

• Bead homogenization with liquid nitrogen
• Adding Buffer AP1 and RNase
• Enzymatic lysis for 30 min
• After adding neutralization buffer, prolonged incubation on ice for 15 min to allow for thorough precipitation of salts
• Centrifugation at 14 000 rpm for 7 min to pellet salts
• Running supernatant through the QIAshredder column
• Adding 1.5 times the lysate volume of AW1 to the lysate
• Running lysate through the column
• Three washing steps with 500 µl Buffer AW2, with 5 min incubation each (last centrifugation of Buffer AW2 at 14 000 rpm for 3 min to avoid ethanol carry-over)
• Elution with 50 µl HPLC H20 after incubation for 1 min
• Second elution with the flow-through

As you notice, the protocol has already been adjusted, but so far, these modifications are not effective. Extractions using the protocol provided with the kit even yield poorer results.

Interestingly, upon adding MagBio High Prep beads to perform a bead clean-up, the solution clumped. Clumps were resuspended through pipetting up and down. However, 260/280 and 260/230 nm ratios of the cleaned extractions were even worse.

Such phenomenon is also described here:

https://twitter.com/amandatron89/status/1048236921615519745?lang=cs

http://seqanswers.com/forums/showthread.php?t=47522

Given that 260/280 nm ratios are okay (little bit elevated in general), it is unlikely that proteins are a problem here and hence, polysaccharides seem to be the culprits. However, I am very happy to discuss and thankful for any advice.