SEA-PHAGES | All posts created by viknesh

Link to this post \| posted 13 Oct, 2018 02:47
viknesh	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 Thanks for pointing that out. The concentrations were only included in the Discovery Guide, and not in the Instructors Guide. It's now been updated.

Link to this post | posted 13 Oct, 2018 02:47

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

Thanks for pointing that out. The concentrations were only included in the Discovery Guide, and not in the Instructors Guide. It's now been updated.

Posted in: Phage Discovery/Isolation → DNA Isolation from M. foliorum - any updates?

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

Posted in: Phage Discovery/Isolation → DNA Isolation from M. foliorum - any updates?

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

Posted in: Phage Discovery/Isolation → DNA Isolation from M. foliorum - any updates?

Link to this post \| posted 10 Sep, 2018 15:39
viknesh	Hi Jake - thanks for writing. The app is down at the moment, and will likely not be back up again for a while. We suggest that you have your students record the information in their lab notebooks, and use their smartphones or computers to determine the GPS location of their collection sites. We've removed instructions for using the app from the Phage Discovery Guide. Edited 10 Sep, 2018 15:41

Posted in: SEA-PHAGES App → a work in progress

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

Posted in: Phage Discovery/Isolation → DNA Isolation from M. foliorum - any updates?

Link to this post \| posted 27 Aug, 2018 18:13
viknesh	kaylafast P2FF streak from stock #1 Incubation: 37 degrees C, 2 days (1 day photo unavailable) I would be suspicious of this streak plate (and the culture from which the streak was setup). There appears to be more growth than expected. Go ahead and test your latest culture, but also setup streak plates (and mock streaks) from your glycerol stocks. Let me know what you see. You can then go ahead and setup cultures.

Link to this post | posted 27 Aug, 2018 18:13

viknesh

kaylafast
P2FF streak from stock #1
Incubation: 37 degrees C, 2 days (1 day photo unavailable)

I would be suspicious of this streak plate (and the culture from which the streak was setup). There appears to be more growth than expected. Go ahead and test your latest culture, but also setup streak plates (and mock streaks) from your glycerol stocks. Let me know what you see. You can then go ahead and setup cultures.

Posted in: Phage Discovery/Isolation → D29 plaques barely visible on M. smeg

Link to this post \| posted 27 Aug, 2018 15:59
viknesh	kaylafast P2FF streak from stock #1 Kayla, can you include the incubation duration before the pictures were taken, for each plate?

Posted in: Phage Discovery/Isolation → D29 plaques barely visible on M. smeg

Link to this post \| posted 27 Aug, 2018 15:43
viknesh	kaylafast I have read the suggestion to streak out the liquid culture and if there are any colonies in 24 hrs, it is not smeg - or the smeg is contaminated. Does this mean any growth at all within 24 hours or just individual colonies? Streaks from our P1FF and P2FF do show growth within 24 hours, but just smears not individual colonies. Hi Kayla, A few things: 1 - Would you be able to share pictures of your streak plates (both from glycerol but also from liquid culture)? You'd expect them to behave similarly, with growth perhaps faster for the plate streaked from the liquid culture. 2 - From the pic you attached, it is hard to tell if a) there is a robust bacterial lawn but turbid plaques, b)light/non-robust lawn with turbid plaques, or c)light/non-robust lawn with clear plaques. If the plaques are turbid, it is likely that you have a contaminating microbe in your liquid culture that is not lysed by D29. When you setup liquid cultures, it maybe worth setting up a mock culture. For this, you do exactly what you do as you are setting up your liquid culture, but instead of picking up a colony with an inoculating stick/loop and dipping in into the liquid media, you just dip a sterile inoculating stick/loop directly into the media. This way you can determine if your flask, media, or inoculating loop is contaminated. Vic

Link to this post | posted 27 Aug, 2018 15:43

viknesh

kaylafast
I have read the suggestion to streak out the liquid culture and if there are any colonies in 24 hrs, it is not smeg - or the smeg is contaminated. Does this mean any growth at all within 24 hours or just individual colonies? Streaks from our P1FF and P2FF do show growth within 24 hours, but just smears not individual colonies.

Hi Kayla,

A few things:

1 - Would you be able to share pictures of your streak plates (both from glycerol but also from liquid culture)? You'd expect them to behave similarly, with growth perhaps faster for the plate streaked from the liquid culture.

2 - From the pic you attached, it is hard to tell if a) there is a robust bacterial lawn but turbid plaques, b)light/non-robust lawn with turbid plaques, or c)light/non-robust lawn with clear plaques. If the plaques are turbid, it is likely that you have a contaminating microbe in your liquid culture that is not lysed by D29. When you setup liquid cultures, it maybe worth setting up a mock culture. For this, you do exactly what you do as you are setting up your liquid culture, but instead of picking up a colony with an inoculating stick/loop and dipping in into the liquid media, you just dip a sterile inoculating stick/loop directly into the media. This way you can determine if your flask, media, or inoculating loop is contaminated.

Vic

Posted in: Phage Discovery/Isolation → D29 plaques barely visible on M. smeg

Link to this post \| posted 07 Jul, 2018 14:53
viknesh	asprenkle Cohort 11, going to the 11B workshop tomorrow! Downloaded the app from Google Play to my android phone. Tried to create account using .edu email and nothing happens. Preparatory info for the workshop suggests that I should get a link confirming the account from my email? Tried restarting my phone, no effect. Any ideas? Hi Amy, The app is down at the moment. When you check-in at UMBC, we'll tell you how to go about sample collection without using the app. See you soon!

Link to this post | posted 07 Jul, 2018 14:53

viknesh

asprenkle
Cohort 11, going to the 11B workshop tomorrow! Downloaded the app from Google Play to my android phone. Tried to create account using .edu email and nothing happens. Preparatory info for the workshop suggests that I should get a link confirming the account from my email?
Tried restarting my phone, no effect. Any ideas?

Hi Amy,

The app is down at the moment. When you check-in at UMBC, we'll tell you how to go about sample collection without using the app. See you soon!

Posted in: SEA-PHAGES App → a work in progress

Link to this post \| posted 10 Nov, 2017 18:24
viknesh	Adaptive Phage Therapeutics (APT) is currently in need of experienced bacteriophage microbiologists to join our team. Attached to this post is a job description for a BS- or MS-level bacteriophage microbiologist. APT is a clinical-stage pharmaceutical company based in Gaithersburg, Maryland that is focused on developing an effective and personalized bacteriophage therapy in response to the global rise of multi-drug resistant (MDR) bacterial pathogens. Two key components to APT's approach include a large and dynamically growing collection of bacteriophages, or PhageBank™, and an innovative rapid system for matching bacteriophage to patient-specific bacterial infections called the Host Range Quick Test. See links below for more information about Adaptive Phage Therapeutics, Inc.: Company Website: http://www.aphage.com News Articles: 1) http://www.businesswire.com/news/home/20170426006880/en/Adaptive-Phage-Therapeutics---Terminally-Ill-Patient 2) https://www.buzzfeed.com/azeenghorayshi/navy-phage-viruses-for-antibiotics-crisis?utm_term=.gvym0OEXQ#.vdmjdPXNV 3) https://www.nbcnews.com/mach/science/post-antibiotic-apocalypse-can-be-prevented-here-s-how-ncna812576

Link to this post | posted 10 Nov, 2017 18:24

viknesh

Adaptive Phage Therapeutics (APT) is currently in need of experienced bacteriophage microbiologists to join our team. Attached to this post is a job description for a BS- or MS-level bacteriophage microbiologist.

APT is a clinical-stage pharmaceutical company based in Gaithersburg, Maryland that is focused on developing an effective and personalized bacteriophage therapy in response to the global rise of multi-drug resistant (MDR) bacterial pathogens. Two key components to APT's approach include a large and dynamically growing collection of bacteriophages, or PhageBank™, and an innovative rapid system for matching bacteriophage to patient-specific bacterial infections called the Host Range Quick Test.

See links below for more information about Adaptive Phage Therapeutics, Inc.:

Company Website: http://www.aphage.com
News Articles:
1) http://www.businesswire.com/news/home/20170426006880/en/Adaptive-Phage-Therapeutics---Terminally-Ill-Patient
2) https://www.buzzfeed.com/azeenghorayshi/navy-phage-viruses-for-antibiotics-crisis?utm_term=.gvym0OEXQ#.vdmjdPXNV
3) https://www.nbcnews.com/mach/science/post-antibiotic-apocalypse-can-be-prevented-here-s-how-ncna812576

Posted in: General Message Board → Bacteriophage Microbiologist Position

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