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 yesterday, 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 yesterday, 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 yesterday, 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 yesterday, 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 yesterday, 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 yesterday, 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 yesterday, 19:34

Link to this post | posted yesterday, 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 yesterday, 19:34

Link to this post \| posted yesterday, 20:54
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 yesterday, 20:56 1.70Mb

Link to this post | posted yesterday, 20:54

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 yesterday, 20:56

Link to this post \| posted yesterday, 21:01
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 yesterday, 21:07 531Kb

Link to this post \| posted yesterday, 22:42
viknesh	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.) Looks like a nice large band. I'm not sure DNA needs to be re-extracted from the phage. Might be worth seeing what the digest looks like.

Link to this post | posted yesterday, 22:42

viknesh

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.)

Looks like a nice large band. I'm not sure DNA needs to be re-extracted from the phage. Might be worth seeing what the digest looks like.

Link to this post \| posted yesterday, 22:46
viknesh	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). HaeIII is a 4-bp cutter, so it is expected to cut more often that a typical 6 bp cutter. Its restriction site is also a GGCC, so a genome with a high GC content will be expected to be restricted often, hence the small bands. When HaeIII cuts often, you can expect the opposite for MseI, which is also a 4 bp cutter but cuts at TTAA – which is what you observed. HaeIII and MseI are therefore a good pair of enzymes to use to get a sense of phage GC content. Edited yesterday, 22:47

Link to this post | posted yesterday, 22:46

viknesh

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).

HaeIII is a 4-bp cutter, so it is expected to cut more often that a typical 6 bp cutter. Its restriction site is also a GGCC, so a genome with a high GC content will be expected to be restricted often, hence the small bands. When HaeIII cuts often, you can expect the opposite for MseI, which is also a 4 bp cutter but cuts at TTAA – which is what you observed. HaeIII and MseI are therefore a good pair of enzymes to use to get a sense of phage GC content.

Edited yesterday, 22:47

Link to this post \| posted 24 minutes ago
cdshaffer	one more point to consider is that that A260/A280 ratio is the average expected when there is a very large number of different proteins. This makes sense when you purify your DNA from a whole cell. But phage have only a small number of proteins which can vary widely in the fraction of Trp, Tyr, and Phe. So the assumptions that underly that A260/A280 may not be valid for any particular phage. If you are worried about sequencing, I would not be. Those gels look pretty good and clearly there is nothing inhibiting restriction digests, so the DNA prep is probably just fine. Anyway there is my magic hand wavy argument for why I tend not to worry about those ratios when doing phage. But it does have a prediction which does allow one to propose an experiment, if you can get this phage sequenced. You could have the students look to see if there are a significantly different number of those critical amino acids in the structural genes for that one phage compared to some of the others which have given more typical ratios. Edited 21 minutes ago

Link to this post | posted 24 minutes ago

cdshaffer

one more point to consider is that that A260/A280 ratio is the average expected when there is a very large number of different proteins. This makes sense when you purify your DNA from a whole cell. But phage have only a small number of proteins which can vary widely in the fraction of Trp, Tyr, and Phe. So the assumptions that underly that A260/A280 may not be valid for any particular phage. If you are worried about sequencing, I would not be. Those gels look pretty good and clearly there is nothing inhibiting restriction digests, so the DNA prep is probably just fine. Anyway there is my magic hand wavy argument for why I tend not to worry about those ratios when doing phage.

But it does have a prediction which does allow one to propose an experiment, if you can get this phage sequenced. You could have the students look to see if there are a significantly different number of those critical amino acids in the structural genes for that one phage compared to some of the others which have given more typical ratios.

Edited 21 minutes ago

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260/280 ratios too high from DNA extraction