SEA-PHAGES | DNA Extraction Troubleshooting - Can we skip the nuclease treatment?

Link to this post \| posted 09 Nov, 2022 19:41
plconnerly	We could use any and all advice about DNA extraction from Gordonia rubripertincta phages. Last year we tried and troubleshot the Wizard DNA Cleanup kit column method with no success. We did phenol:chloroform:isoamyl alcohol as a last resort and managed to get useable DNA from 1 phage. This year we're trying the ZnCl2 method and initial runs have not worked. We've got some troubleshooting planed and one idea we have is to leave out the initial nuclease step. Is that allowed? Have other folks tried that? Can DNA extracted without nuclease treatment be used for sequencing? Thanks! Pam - IU Southeast

Link to this post | posted 09 Nov, 2022 19:41

We could use any and all advice about DNA extraction from Gordonia rubripertincta phages. Last year we tried and troubleshot the Wizard DNA Cleanup kit column method with no success. We did phenol:chloroform:isoamyl alcohol as a last resort and managed to get useable DNA from 1 phage. This year we're trying the ZnCl2 method and initial runs have not worked. We've got some troubleshooting planed and one idea we have is to leave out the initial nuclease step. Is that allowed? Have other folks tried that? Can DNA extracted without nuclease treatment be used for sequencing?
Thanks!
Pam - IU Southeast

Link to this post \| posted 09 Nov, 2022 19:46
viknesh	plconnerly We could use any and all advice about DNA extraction from Gordonia rubripertincta phages. Last year we tried and troubleshot the Wizard DNA Cleanup kit column method with no success. We did phenol:chloroform:isoamyl alcohol as a last resort and managed to get useable DNA from 1 phage. This year we're trying the ZnCl2 method and initial runs have not worked. We've got some troubleshooting planed and one idea we have is to leave out the initial nuclease step. Is that allowed? Have other folks tried that? Can DNA extracted without nuclease treatment be used for sequencing? Thanks! Pam - IU Southeast I'm hoping Dan Russell will chime in here but I believe it is very important to include the nucelase treatment step. Otherwise, the bacterial DNA to phage DNA ratio might be too high, resulting in insufficient sequencing depth for the phage genome. Can I ask about the titer of the samples that are resulting is low DNA extraction?

Link to this post | posted 09 Nov, 2022 19:46

viknesh

plconnerly
We could use any and all advice about DNA extraction from Gordonia rubripertincta phages. Last year we tried and troubleshot the Wizard DNA Cleanup kit column method with no success. We did phenol:chloroform:isoamyl alcohol as a last resort and managed to get useable DNA from 1 phage. This year we're trying the ZnCl2 method and initial runs have not worked. We've got some troubleshooting planed and one idea we have is to leave out the initial nuclease step. Is that allowed? Have other folks tried that? Can DNA extracted without nuclease treatment be used for sequencing?
Thanks!
Pam - IU Southeast

I'm hoping Dan Russell will chime in here but I believe it is very important to include the nucelase treatment step. Otherwise, the bacterial DNA to phage DNA ratio might be too high, resulting in insufficient sequencing depth for the phage genome.

Can I ask about the titer of the samples that are resulting is low DNA extraction?

Link to this post \| posted 09 Nov, 2022 21:13
plconnerly	Vic - we're using all samples with a titer of at least 1 x 10^9.

Link to this post \| posted 10 Nov, 2022 14:55
jwertz	This year we are using the protocol described in the attached manuscript. We had students use 900 ul of HVL to start, splitting that into two spin columns and eluting each column twice. Most results were a bit on the low side, but several got sequencable amounts. We are using Rhodococcus equi as a host this year. 562Kb

Link to this post \| posted 10 Nov, 2022 16:33
viknesh	plconnerly Vic - we're using all samples with a titer of at least 1 x 10^9. Typically, we can get the minimum 40 ng/ul from a lysate at 5x10^9 pfu/ml, though of course that will vary from phage to phage. Including a ZnCl2 precipitation step has worked well for many, with University of Ottawa now including this as a standard step for all their DNA extractions. It does take a little getting used to, and it is very important to be resuspending the pellet very quickly after the spin, and in EDTA. The precipitation step is harsh and phage DNA is rapidly released after the spin, making them accessible to the nuclease before the denaturant is added. I havent tried this, but perhaps it makes sense to add EDTA to the nuclease-treated lysate before adding ZnCl2. As you troubleshoot, I'd recommend prepping DNA from one lysate both ways (with and without ZnCl2), side by side, to see if the precipitation step works for you. Let me know if you want to chat via Zoom.

Link to this post | posted 10 Nov, 2022 16:33

viknesh

plconnerly
Vic - we're using all samples with a titer of at least 1 x 10^9.

Typically, we can get the minimum 40 ng/ul from a lysate at 5x10^9 pfu/ml, though of course that will vary from phage to phage. Including a ZnCl2 precipitation step has worked well for many, with University of Ottawa now including this as a standard step for all their DNA extractions. It does take a little getting used to, and it is very important to be resuspending the pellet very quickly after the spin, and in EDTA. The precipitation step is harsh and phage DNA is rapidly released after the spin, making them accessible to the nuclease before the denaturant is added. I havent tried this, but perhaps it makes sense to add EDTA to the nuclease-treated lysate before adding ZnCl2.

As you troubleshoot, I'd recommend prepping DNA from one lysate both ways (with and without ZnCl2), side by side, to see if the precipitation step works for you.

Let me know if you want to chat via Zoom.

Link to this post \| posted 12 Nov, 2022 19:21
mdgainey	I have had good luck with some but not all of my student's G. rubri phages by taking 4mL of the students HTL pretreating with DNase/RNase, then spinning for 10,000rpm for 40 min at 4 degrees C as described in the instructors guide, and pulling off enough of the supernatant to end up with ~500ul of the end. I then immediately add EDTA ect. It says in the guide that this method is not as consistent as the ZnCl2 protocol, which may be true but it is easy and normally bumps up a few of my students DNA concentrations nicely without me having to have the students make a bunch of new stocks. I would be happy to send you the full protocol I use if you like. You could also do the spin, and then try phenol/chloroform extraction, instead of the wizard kit if you are comfortable with that.

Link to this post | posted 12 Nov, 2022 19:21

mdgainey

I have had good luck with some but not all of my student's G. rubri phages by taking 4mL of the students HTL pretreating with DNase/RNase, then spinning for 10,000rpm for 40 min at 4 degrees C as described in the instructors guide, and pulling off enough of the supernatant to end up with ~500ul of the end. I then immediately add EDTA ect. It says in the guide that this method is not as consistent as the ZnCl2 protocol, which may be true but it is easy and normally bumps up a few of my students DNA concentrations nicely without me having to have the students make a bunch of new stocks. I would be happy to send you the full protocol I use if you like. You could also do the spin, and then try phenol/chloroform extraction, instead of the wizard kit if you are comfortable with that.

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DNA Extraction Troubleshooting - Can we skip the nuclease treatment?