SEA-PHAGES | DNA Isolation from M. foliorum

Link to this post \| posted 07 Sep, 2018 14:34
MSMC	I had discussions with multiple people at the symposium and faculty retreat about issues with extracting DNA from M. foliorum phages. It seemed that many people were running into an issue with increased smearing of the phage DNA when the digests were run on a gel. Has anyone tried any modifications to the protocol? We did one pilot where we omitted DNase, it did not have a large effect (only tried once though). I was thinking of increasing the time of guanadinium thiocyanate exposure a bit. Any thoughts?

Link to this post | posted 07 Sep, 2018 14:34

I had discussions with multiple people at the symposium and faculty retreat about issues with extracting DNA from M. foliorum phages. It seemed that many people were running into an issue with increased smearing of the phage DNA when the digests were run on a gel. Has anyone tried any modifications to the protocol? We did one pilot where we omitted DNase, it did not have a large effect (only tried once though). I was thinking of increasing the time of guanadinium thiocyanate exposure a bit. Any thoughts?

Link to this post \| posted 08 Sep, 2018 02:16
viknesh	Hi Evan, We've posted a modification that we know works for extracting DNA from M. foliorum phages. The modification is listed as an optional step in the DNA extraction protocol, and involves adding ProteinaseK and SDS. Vic

Link to this post \| posted 10 Sep, 2018 00:09
MSMC	Thanks Vic!

Link to this post \| posted 12 Oct, 2018 14:38
c.sunnen	Vic, Is the suggestion to use ProteinaseK and SDS instead of EDTA, or to use both? This is our first go with M. foliorum. Also, we'll be using the ZnCl method. Have either of these nuclease inhibition steps been tried with the ZnCl method? I'm attaching our current protocol (modified to include an EDTA step; this is the only change from how we did it with smeg last year), in case that's useful. Thanks all! I love this community! Nikki 16Kb

Link to this post | posted 12 Oct, 2018 14:38

c.sunnen

Vic,

Is the suggestion to use ProteinaseK and SDS instead of EDTA, or to use both? This is our first go with M. foliorum. Also, we'll be using the ZnCl method. Have either of these nuclease inhibition steps been tried with the ZnCl method? I'm attaching our current protocol (modified to include an EDTA step; this is the only change from how we did it with smeg last year), in case that's useful.

Thanks all! I love this community!
Nikki

Link to this post \| posted 12 Oct, 2018 15:22
viknesh	c.sunnen Vic, Is the suggestion to use ProteinaseK and SDS instead of EDTA, or to use both? This is our first go with M. foliorum. Also, we'll be using the ZnCl method. Have either of these nuclease inhibition steps been tried with the ZnCl method? I'm attaching our current protocol (modified to include an EDTA step; this is the only change from how we did it with smeg last year), in case that's useful. Thanks all! I love this community! Nikki Hi Nikki, A couple of things. For M. foliorum, it is important to use ProtK and SDS to remove residual nuclease activity from DNA preps. Residual nuclease is not a problem during the prep process itself, since EDTA is present. Once eluted in water, the lack of EDTA is still not a problem since the nucleases have no access to metal cofactors required for activity. However, any downstream analysis using buffers will activate the nucleases. If you can get away with the standard DNA extraction protocol (wihtout a precipitation step), I would encourage that because we've tested that protocol (with ProtK and SDS) extensively over the summer. We have some, but significantly less, experience using ProtK with the zinc precipitation protocol. If you do decide to continue with the zinc chloride precipitation protocol, here are some things to note. With the zinc chloride protocol, EDTA has to be added after the precipitation step, since EDTA will chelate the zinc that is needed for the precipitation step. Currently, the Instructors Guide suggests the following: Add nuclease to the lysate -> precipitate with zinc chloride -> resuspend in EDTA -> add ProtK + SDS to the lysate -> continue with standard prep. One issue with this workflow is that the precipitation step results in a significant fraction of particles releasing DNA during the resuspension step. It is therefore critical that EDTA be used for resuspension, and that you work quickly to add the EDTA. Otherwise, any residual nuclease will rapidly degrade DNA that is released during pellet resuspension. In the Instructors Guide, I recommed using the "alternative" strategy for Step D1. Once resuspended in EDTA, you can then add ProtK and SDS to remove residual nuclease. Another workflow would be as follows: nuclease-treat the lysate -> add ProtK + SDS to the lysate -> precipitate with ZnCl -> resuspend in EDTA -> continue with standard prep. I've not tested this workflow. The hope here is that ProtK will remove all nuclease activity prior to precipitation, allowing you to work more slowly during the resuspension step. However, my fear here is that SDS might destabilize some fraction of phages, in a phage-dependent manner, and you'll end up losing a significant fraction of DNA to the supernatant during the precipitation step. If you have a student that has extra time and wants to help test this, let me know Vic Edited 12 Oct, 2018 15:27

Link to this post | posted 12 Oct, 2018 15:22

viknesh

c.sunnen
Vic,

Is the suggestion to use ProteinaseK and SDS instead of EDTA, or to use both? This is our first go with M. foliorum. Also, we'll be using the ZnCl method. Have either of these nuclease inhibition steps been tried with the ZnCl method? I'm attaching our current protocol (modified to include an EDTA step; this is the only change from how we did it with smeg last year), in case that's useful.

Thanks all! I love this community!
Nikki

Hi Nikki,

A couple of things. For M. foliorum, it is important to use ProtK and SDS to remove residual nuclease activity from DNA preps. Residual nuclease is not a problem during the prep process itself, since EDTA is present. Once eluted in water, the lack of EDTA is still not a problem since the nucleases have no access to metal cofactors required for activity. However, any downstream analysis using buffers will activate the nucleases.

If you can get away with the standard DNA extraction protocol (wihtout a precipitation step), I would encourage that because we've tested that protocol (with ProtK and SDS) extensively over the summer. We have some, but significantly less, experience using ProtK with the zinc precipitation protocol. If you do decide to continue with the zinc chloride precipitation protocol, here are some things to note.

With the zinc chloride protocol, EDTA has to be added after the precipitation step, since EDTA will chelate the zinc that is needed for the precipitation step.

Currently, the Instructors Guide suggests the following:
Add nuclease to the lysate -> precipitate with zinc chloride -> resuspend in EDTA -> add ProtK + SDS to the lysate -> continue with standard prep.
One issue with this workflow is that the precipitation step results in a significant fraction of particles releasing DNA during the resuspension step. It is therefore critical that EDTA be used for resuspension, and that you work quickly to add the EDTA. Otherwise, any residual nuclease will rapidly degrade DNA that is released during pellet resuspension. In the Instructors Guide, I recommed using the "alternative" strategy for Step D1. Once resuspended in EDTA, you can then add ProtK and SDS to remove residual nuclease.

Another workflow would be as follows:
nuclease-treat the lysate -> add ProtK + SDS to the lysate -> precipitate with ZnCl -> resuspend in EDTA -> continue with standard prep.
I've not tested this workflow. The hope here is that ProtK will remove all nuclease activity prior to precipitation, allowing you to work more slowly during the resuspension step. However, my fear here is that SDS might destabilize some fraction of phages, in a phage-dependent manner, and you'll end up losing a significant fraction of DNA to the supernatant during the precipitation step. If you have a student that has extra time and wants to help test this, let me know smile

Vic

Edited 12 Oct, 2018 15:27

Link to this post \| posted 12 Oct, 2018 17:09
c.sunnen	Vic, This is incredibly helpful - thank-you! And I'm so glad that you pointed out how we can't add the EDTA before the ZnCl (of course, but I didn't catch it!). Also, I didn't see that a version of this protocol was available in the updated instructor's guide, so now we can see how closely our current version matches! We plan to do a "practice" run with our students next week with small volumes of phage lysate (we find that they always mess up the first time they encounter any DNA isolation protocol, and end up wasting a bunch of precious lysate and reagents), so this will be a good opportunity to try the first workflow you suggested. I'll stress that speed is important when adding EDTA, and hope for the best! Clarification question: I know in the instructor's guide protocol you switch to using the Wizard kit at the capsid denaturation step, whereas traditionally (by our ZnCl protocol) we would do this in TES buffer (0.1M Tris-HCl, pH 8, 0.1M EDTA, 0.3% SDS). Since we don't intend to use the Wizard kit at all, would you recommend eliminating the TES step and just proceeding to the potassium acetate precipitation? It seems that if we're resuspending in EDTA instead of TES, the composition would be very similar, and I'm assuming accomplish the same thing: EDTA concentration would be the same, and then SDS would be added with the proteinase K… What %SDS should be added with the protK? Thank-you again! I'll be sure to let you know how it goes! Nikki Edited 12 Oct, 2018 17:39

Link to this post | posted 12 Oct, 2018 17:09

c.sunnen

Vic,

This is incredibly helpful - thank-you! And I'm so glad that you pointed out how we can't add the EDTA before the ZnCl (of course, but I didn't catch it!). Also, I didn't see that a version of this protocol was available in the updated instructor's guide, so now we can see how closely our current version matches!

We plan to do a "practice" run with our students next week with small volumes of phage lysate (we find that they always mess up the first time they encounter any DNA isolation protocol, and end up wasting a bunch of precious lysate and reagents), so this will be a good opportunity to try the first workflow you suggested. I'll stress that speed is important when adding EDTA, and hope for the best!

Clarification question: I know in the instructor's guide protocol you switch to using the Wizard kit at the capsid denaturation step, whereas traditionally (by our ZnCl protocol) we would do this in TES buffer (0.1M Tris-HCl, pH 8, 0.1M EDTA, 0.3% SDS). Since we don't intend to use the Wizard kit at all, would you recommend eliminating the TES step and just proceeding to the potassium acetate precipitation? It seems that if we're resuspending in EDTA instead of TES, the composition would be very similar, and I'm assuming accomplish the same thing: EDTA concentration would be the same, and then SDS would be added with the proteinase K… What %SDS should be added with the protK?

Thank-you again!

I'll be sure to let you know how it goes!
Nikki

Edited 12 Oct, 2018 17:39

Link to this post \| posted 12 Oct, 2018 17:50
viknesh	c.sunnen Clarification question: I know in the instructor's guide protocol you switch to using the Wizard kit at the capsid denaturation step, whereas traditionally (by our ZnCl protocol) we would do this in TES buffer (0.1M Tris-HCl, pH 8, 0.1M EDTA, 0.3% SDS). Since we don't intend to use the Wizard kit at all, would you recommend eliminating the TES step and just proceeding to the potassium acetate precipitation? It seems that if we're resuspending in EDTA instead of TES, the composition would be very similar, and I'm assuming accomplish the same thing: EDTA concentration would be the same, and then SDS would be added with the proteinase K… What %SDS should be added with the protK? Ah, I now see that you intend to complete the protocol by precipitating DNA instead of using the Wizard columns. You are correct - you can go ahead and resuspend in TES, then add ProtK. In the zinc chloride precipitation protocol, we use EDTA instead of TES just to make things easier for those wanting to precipitate with zinc and continue with the old protocol (that is, they do not have to make TES). However, considering that you will use both zinc and protK, and therefore EDTA and SDS, using TES makes sense. The conc. of SDS recommended for ProtK is 0.5% w/v. Perhaps you could prepare your TES to contain 0.1M EDTA and 0.5% SDS. Edited 14 Oct, 2018 16:31

Link to this post | posted 12 Oct, 2018 17:50

viknesh

c.sunnen
Clarification question: I know in the instructor's guide protocol you switch to using the Wizard kit at the capsid denaturation step, whereas traditionally (by our ZnCl protocol) we would do this in TES buffer (0.1M Tris-HCl, pH 8, 0.1M EDTA, 0.3% SDS). Since we don't intend to use the Wizard kit at all, would you recommend eliminating the TES step and just proceeding to the potassium acetate precipitation? It seems that if we're resuspending in EDTA instead of TES, the composition would be very similar, and I'm assuming accomplish the same thing: EDTA concentration would be the same, and then SDS would be added with the proteinase K… What %SDS should be added with the protK?

Ah, I now see that you intend to complete the protocol by precipitating DNA instead of using the Wizard columns. You are correct - you can go ahead and resuspend in TES, then add ProtK. In the zinc chloride precipitation protocol, we use EDTA instead of TES just to make things easier for those wanting to precipitate with zinc and continue with the old protocol (that is, they do not have to make TES). However, considering that you will use both zinc and protK, and therefore EDTA and SDS, using TES makes sense. The conc. of SDS recommended for ProtK is 0.5% w/v. Perhaps you could prepare your TES to contain 0.1M EDTA and 0.5% SDS.

Edited 14 Oct, 2018 16:31

Link to this post \| posted 12 Oct, 2018 19:18
c.sunnen	Thank-you, thank-you, thank-you!!!

Link to this post \| posted 12 Oct, 2018 22:22
mdgainey	Hi I thought I would just add a bit to this thread since I had some issues with my preps last year. For our initial digest analysis this year I think I am just going to omit the DNase and RNase treatment altogether, and instead of precipitating the DNA I am going to do the 4 degree concentration spin that the guide suggests, and then prep/digest. I had good luck with this, this summer with some of my Microphages that I had issues with last semester and got nice clean yields. For the phages selected for sequencing, I will use the protocol as Vic has suggested. Just thought I would throw this out there as another option to try.

Link to this post | posted 12 Oct, 2018 22:22

mdgainey

Hi I thought I would just add a bit to this thread since I had some issues with my preps last year. For our initial digest analysis this year I think I am just going to omit the DNase and RNase treatment altogether, and instead of precipitating the DNA I am going to do the 4 degree concentration spin that the guide suggests, and then prep/digest. I had good luck with this, this summer with some of my Microphages that I had issues with last semester and got nice clean yields. For the phages selected for sequencing, I will use the protocol as Vic has suggested. Just thought I would throw this out there as another option to try.

Link to this post \| posted 13 Oct, 2018 00:01
c.sunnen	Sorry to keep bugging, but I'm revising our protocol: what's the concentration of Proteinase K? The instructor guide protocol calls for 5uL per 1mL sample, but it doesn't have a concentration. Also, you recommended above 0.5M EDTA, but the protocol says 0.1M. Is one concentration preferred. Thank-you again for all of your input, and thanks Maria for your insight! If we have trouble, we'll probably eliminate the nuclease step, too! Nikki

Link to this post | posted 13 Oct, 2018 00:01

c.sunnen

Sorry to keep bugging, but I'm revising our protocol: what's the concentration of Proteinase K? The instructor guide protocol calls for 5uL per 1mL sample, but it doesn't have a concentration. Also, you recommended above 0.5M EDTA, but the protocol says 0.1M. Is one concentration preferred.

Thank-you again for all of your input, and thanks Maria for your insight! If we have trouble, we'll probably eliminate the nuclease step, too!

Nikki

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