SEA-PHAGES | 260/280 ratios too high from DNA extraction

Link to this post \| posted 19 Jul, 2022 18:18
MSMC	We are using the Wizard DNA Clean-Up System kit for DNA extraction (as we have for many years), and recently our DNA samples have had extremely high 260/280 ratios (most between 2.6 to 3.0). Things we've tried: 1. New Kit (Guanididium and Columns) 2. New Water Eluent 3. New EDTA 4. New SDS 5. Syringe vs. Vacuum manifold Our spec seems fine, as other samples give normal ratios. The A260 seems high (vs. low 280), as when we run samples on the gel, the mass appears lower on the gel than calculated by the spec. We're running out of ideas, any thoughts?

Link to this post | posted 19 Jul, 2022 18:18

We are using the Wizard DNA Clean-Up System kit for DNA extraction (as we have for many years), and recently our DNA samples have had extremely high 260/280 ratios (most between 2.6 to 3.0). Things we've tried:

1. New Kit (Guanididium and Columns)
2. New Water Eluent
3. New EDTA
4. New SDS
5. Syringe vs. Vacuum manifold

Our spec seems fine, as other samples give normal ratios. The A260 seems high (vs. low 280), as when we run samples on the gel, the mass appears lower on the gel than calculated by the spec.

We're running out of ideas, any thoughts?

Link to this post \| posted today, 01:21
lmeadows@rcbc.edu	One of our six phage DNA extracts is showing us the same thing. Did you ever figure out the issue? All of our other samples look good.

Link to this post \| posted today, 17:21
nic.vega	It might be guanidine carryover - the stuff is hard to get rid of. Make sure you're using fresh stocks of isopropanol & EtOH, whichever is relevant. If you have the facilities to do so, you can try re-precipitating in EtOH and washing the existing DNA samples again (something like https://barricklab.org/twiki/bin/view/Lab/ProtocolsEthanolPrecipitation) which should help a lot to clear any remaining salts.

Link to this post | posted today, 17:21

nic.vega

It might be guanidine carryover - the stuff is hard to get rid of. Make sure you're using fresh stocks of isopropanol & EtOH, whichever is relevant. If you have the facilities to do so, you can try re-precipitating in EtOH and washing the existing DNA samples again (something like https://barricklab.org/twiki/bin/view/Lab/ProtocolsEthanolPrecipitation) which should help a lot to clear any remaining salts.

Link to this post \| posted today, 19:02
lmeadows@rcbc.edu	Thanks for the quick reply. I thought guanidine carryover caused high DNA concentration estimates from the Nanopore, but caused low A260/280 ratios (instead of high ratios). The only thing I could find online that caused high ratios was RNA contamination, but we are not seeing that in our other samples. I add the nucleases to the student samples during the DNA purification process, so I know this student had it added to her sample. But both times, her A260/280 ratios were above 2.2.

Link to this post | posted today, 19:02

lmeadows@rcbc.edu

Thanks for the quick reply. I thought guanidine carryover caused high DNA concentration estimates from the Nanopore, but caused low A260/280 ratios (instead of high ratios). The only thing I could find online that caused high ratios was RNA contamination, but we are not seeing that in our other samples. I add the nucleases to the student samples during the DNA purification process, so I know this student had it added to her sample. But both times, her A260/280 ratios were above 2.2.

Link to this post \| posted today, 19:33
viknesh	lmeadows@rcbc.edu Thanks for the quick reply. I thought guanidine carryover caused high DNA concentration estimates from the Nanopore, but caused low A260/280 ratios (instead of high ratios). The only thing I could find online that caused high ratios was RNA contamination, but we are not seeing that in our other samples. I add the nucleases to the student samples during the DNA purification process, so I know this student had it added to her sample. But both times, her A260/280 ratios were above 2.2. If you have run the sample on a gel, could you share the gel image? If not, you might want to consider doing so. If your RNase A is not working, you'll see that RNA contamination in the gel. If it is only RNA, that might be exciting too!!! Edited today, 19:34

Link to this post | posted today, 19:33

viknesh

lmeadows@rcbc.edu
Thanks for the quick reply. I thought guanidine carryover caused high DNA concentration estimates from the Nanopore, but caused low A260/280 ratios (instead of high ratios). The only thing I could find online that caused high ratios was RNA contamination, but we are not seeing that in our other samples. I add the nucleases to the student samples during the DNA purification process, so I know this student had it added to her sample. But both times, her A260/280 ratios were above 2.2.

If you have run the sample on a gel, could you share the gel image? If not, you might want to consider doing so. If your RNase A is not working, you'll see that RNA contamination in the gel. If it is only RNA, that might be exciting too!!!

Edited today, 19:34

Link to this post \| posted 24 minutes ago
lmeadows@rcbc.edu	I'm a bit embarrassed to show this gel – this was a "quick and dirty" - make sure there is actually some DNA - gel that was run after the initial DNA extractions. The student with the problem sample will run another gel with RE digests tomorrow on her second DNA extraction. (The same nuclease mix was used in all six samples, so the RNAse A appears to be working.) Edited 21 minutes ago 1.70Mb

Link to this post \| posted 16 minutes ago
lmeadows@rcbc.edu	Here's a pretty gel from a different sample (to save face) In other random news, HaeIII appeared to completely degrade all six of our phage DNA samples (as shown on this gel) – maybe a problem with that lot number (or I grabbed the wrong tube). Edited 11 minutes ago 531Kb

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260/280 ratios too high from DNA extraction