SEA-PHAGES | All posts created by viknesh

Link to this post \| posted 10 Dec, 2024 19:21
viknesh	nic.vega We did some side-by-side reactions on TrixiePhattel; results attached. The gel was much better this time - it turns out the reason that the first gel was blurry is that the 10X TBE in the teaching lab expired in 2012. Thanks for sharing this – very nice gel. If your student is willing to do it one more time with a third setup: Regular 1 hr; Regular 1 min, MWV 1 min, that'd be helpful. Even in our regular setup (no microwave),I think it is likely that the majority of DNA is restricted within the first minute, which might potentially give us the same results as the MWV reaction. Thanks, Nic. Vic

Link to this post | posted 10 Dec, 2024 19:21

nic.vega
We did some side-by-side reactions on TrixiePhattel; results attached. The gel was much better this time - it turns out the reason that the first gel was blurry is that the 10X TBE in the teaching lab expired in 2012.

Thanks for sharing this – very nice gel. If your student is willing to do it one more time with a third setup: Regular 1 hr; Regular 1 min, MWV 1 min, that'd be helpful. Even in our regular setup (no microwave),I think it is likely that the majority of DNA is restricted within the first minute, which might potentially give us the same results as the MWV reaction.

Thanks, Nic.
Vic

Posted in: Phage Discovery/Isolation → did you know you can do restriction digests in the microwave?

Link to this post \| posted 05 Dec, 2024 22:02
viknesh	Hi Nic, It might be useful to gather more side-by-side data for digests done using our standard method vs the microwave method. It might be quicker to use the microwave, but it’s unclear to me if the quality of the data is where we need it to be (I appreciate that the gel might just be a litle smeary). At the moment, we are beginning to look for phage DNA modifications, which can be gleaned by looking for inconsistencies in the expected digest pattern (i.e. a virtual digest based on the DNA sequence) vs pattern observed when the actual digest is run on a gel. If it is tricky to get good digests and gels using the microwave method, then that’ll impact the utility of the data for that purpose. The TrixiePhatell gel from a regular digest can be viewed here. We’re always excited to hear of new ways of doing our collective research. As we learn of new strategies though, the SEA team usually invests time to understand the implications of the new technique for our collective work before we incorporate anything into our regular workflow. This is so we can continue to leverage the large datasets that are generated by faculty and students from across the program to glean new insights. If some of that data is generated differently, it can impact our interpretation. So if you are willing to gather some data to be reviewed, perhaps over the next semester, please do share. Best, Vic

Link to this post | posted 05 Dec, 2024 22:02

viknesh

Hi Nic,

It might be useful to gather more side-by-side data for digests done using our standard method vs the microwave method. It might be quicker to use the microwave, but it’s unclear to me if the quality of the data is where we need it to be (I appreciate that the gel might just be a litle smeary). At the moment, we are beginning to look for phage DNA modifications, which can be gleaned by looking for inconsistencies in the expected digest pattern (i.e. a virtual digest based on the DNA sequence) vs pattern observed when the actual digest is run on a gel. If it is tricky to get good digests and gels using the microwave method, then that’ll impact the utility of the data for that purpose. The TrixiePhatell gel from a regular digest can be viewed here.

We’re always excited to hear of new ways of doing our collective research. As we learn of new strategies though, the SEA team usually invests time to understand the implications of the new technique for our collective work before we incorporate anything into our regular workflow. This is so we can continue to leverage the large datasets that are generated by faculty and students from across the program to glean new insights. If some of that data is generated differently, it can impact our interpretation. So if you are willing to gather some data to be reviewed, perhaps over the next semester, please do share.

Best,
Vic

Posted in: Phage Discovery/Isolation → did you know you can do restriction digests in the microwave?

Link to this post \| posted 19 Nov, 2024 18:34
viknesh	Click here to download a template for contributing readily-intepretable data that is publishable on QUBES for efforts to raise lysogens for a given phage, regardless if you were successful or unsuccessful at raising lysogens. The datacard template shows the series of experiments that must be conducted (Protocols 11.1, 11.3, and 11.4) and resulting experimental data that must be included in order for your data to reviewed and considered for publication on QUBES. If you have any questions about raising lysogens and publishing your data, please do not hesitate to ask using the form below or emailing the SEA Team. Edited 19 Nov, 2024 19:49

Link to this post | posted 19 Nov, 2024 18:34

viknesh

Click here to download a template for contributing readily-intepretable data that is publishable on QUBES for efforts to raise lysogens for a given phage, regardless if you were successful or unsuccessful at raising lysogens.

The datacard template shows the series of experiments that must be conducted (Protocols 11.1, 11.3, and 11.4) and resulting experimental data that must be included in order for your data to reviewed and considered for publication on QUBES. If you have any questions about raising lysogens and publishing your data, please do not hesitate to ask using the form below or emailing the SEA Team.

Edited 19 Nov, 2024 19:49

Posted in: Lysogeny/Immunity → Recording Data for Attempts to Raise Lysogens

Link to this post \| posted 25 Oct, 2024 02:37
viknesh	rocky2 Hi, I purchased 2 brand new Promega Wizard DNA Cleanup Kit. After warming up, both kits' resins had this black seed/mold-like stuff at the bottom. Is this normal? I added a picture here. Best, Rocky I'e never seen that before, Rocky. I'd be surprised if that was microbial growth since that solution is a very strong denaturant. In any case, it might be worth returning it.

Posted in: Phage Discovery/Isolation → Wizard DNA Cleanup Kit - Resin Issue

Link to this post \| posted 17 Oct, 2024 13:56
viknesh	DrCatalase Hello All, This is our first time using A.g. and we got good DNA extraction using the basic protocol from the discovery guide. However, when we tried digestion with NspI and SacII there were no DNA fragments just the large band of uncut DNA. Is there any trick or should I just try a longer digestion time? Thank you! Sean Hi Sean, Are you seeing this for one phage or for multiple phages from your class? It's not uncommon for a 6-bp cutter like NspI and SacII to not cut phage DNA at all. In fact, this was true for several phages isolated by faculty using A. globiformis at the summer training this year. For that reason, we encourage using a 4-bp cutter. Typically, we recommend using HaeIII, which almost always cuts DNA, which reassures you that your reactions were setup correctly. Thought much less common, if phage DNA is modified, say at the C, then even HaeIII might not cut DNA. To help us decipher such interesting results, more recently, we've encouraged faculty to also include a second 4-bp cutter, MseI; HaeIII and MseI are GC and AT cutters respectively. I dont think longer incubation times are necessary. Instead, I'd recommend two things. First, include HaeIII (and MseI too, if you can). Second, maybe test your NspI and SacII on any DNA that you know they will cut (eg. NspI will cut pUC19 plasmid DNA).

Link to this post | posted 17 Oct, 2024 13:56

viknesh

DrCatalase
Hello All,
This is our first time using A.g. and we got good DNA extraction using the basic protocol from the discovery guide. However, when we tried digestion with NspI and SacII there were no DNA fragments just the large band of uncut DNA. Is there any trick or should I just try a longer digestion time?
Thank you!
Sean

Hi Sean,

Are you seeing this for one phage or for multiple phages from your class?

It's not uncommon for a 6-bp cutter like NspI and SacII to not cut phage DNA at all. In fact, this was true for several phages isolated by faculty using A. globiformis at the summer training this year. For that reason, we encourage using a 4-bp cutter. Typically, we recommend using HaeIII, which almost always cuts DNA, which reassures you that your reactions were setup correctly. Thought much less common, if phage DNA is modified, say at the C, then even HaeIII might not cut DNA. To help us decipher such interesting results, more recently, we've encouraged faculty to also include a second 4-bp cutter, MseI; HaeIII and MseI are GC and AT cutters respectively.

I dont think longer incubation times are necessary. Instead, I'd recommend two things. First, include HaeIII (and MseI too, if you can). Second, maybe test your NspI and SacII on any DNA that you know they will cut (eg. NspI will cut pUC19 plasmid DNA).

Posted in: Phage Discovery/Isolation → A. globiformis Digest Issues?

Link to this post \| posted 30 Sep, 2024 17:11
viknesh	nietof Hi everyone Has anybody successfully isolated phages from soils stored frozen after sampling? Thank you Fernando Wanted to share that no faculty has shared with me that they've tried this.

Posted in: Phage Discovery/Isolation → Isolating from frozen soils

Link to this post \| posted 20 Sep, 2024 14:48
viknesh	bavina Hello, I am also having issues with my PYCa media. I made a batch of broth and top agar and they both became cloudy after adding the CaCl2 solution. There aren't any huge flaky crystals but still a visible change the media almost immediately after adding 1M CaCl2. I waited until the solutions were cooled to 55 C but this still happened. Is this media unusable or can it still be used for experiments? Is the only fix switching to Bacto Peptone like described in the earlier posts? The precipitation could be problematic for at least two reasons: First, it is hard to tell if the media is contaminated and second, the precipitate itself, say calcium chloride, might have been a necessary component of the solution needed for infectivity. If you keep those two things in mind, you can still use your media. To address the possibility of contamonation, you'll want to include controls. For example, when using the top agar for a plaque assay, include a control where no host is added to the top agar. No lawn should form and the top agar should remain clear after 24 hours. To address the concern that precipitation might negatively impact infection, you'll want to pay attention to any change in infectivity for phages that you've isolated. In the longer term, it would be good to change the media components. As we look to no longer use Fiher brand peptone, the HHMI SEA team hasn't had a chance to test alternatives to Bacto products that we know are quite expensive, but we plan to do so – more soon!

Link to this post | posted 20 Sep, 2024 14:48

viknesh

bavina
Hello,

I am also having issues with my PYCa media. I made a batch of broth and top agar and they both became cloudy after adding the CaCl2 solution. There aren't any huge flaky crystals but still a visible change the media almost immediately after adding 1M CaCl2. I waited until the solutions were cooled to 55 C but this still happened. Is this media unusable or can it still be used for experiments? Is the only fix switching to Bacto Peptone like described in the earlier posts?

The precipitation could be problematic for at least two reasons: First, it is hard to tell if the media is contaminated and second, the precipitate itself, say calcium chloride, might have been a necessary component of the solution needed for infectivity. If you keep those two things in mind, you can still use your media. To address the possibility of contamonation, you'll want to include controls. For example, when using the top agar for a plaque assay, include a control where no host is added to the top agar. No lawn should form and the top agar should remain clear after 24 hours. To address the concern that precipitation might negatively impact infection, you'll want to pay attention to any change in infectivity for phages that you've isolated.

In the longer term, it would be good to change the media components. As we look to no longer use Fiher brand peptone, the HHMI SEA team hasn't had a chance to test alternatives to Bacto products that we know are quite expensive, but we plan to do so – more soon!

Posted in: Phage Discovery/Isolation → Precipitation of CaCl2 in PYCA top-agar

Link to this post \| posted 06 Sep, 2024 23:42
viknesh	serivas Hello all, Could "Bacto" Tryptone (pancreatic digest of casein - microbiological peptone) be used to replace the "fisher" brand peptone we are currently using for our top agar? It is likley that you'll be able to culture the bacteria we use for phage-hunting just as well using peptone or tryptone. However, for us to compare data from across the SEA program, I would recommend using peptone. If you use tryptone, it will be important for you to disclose this information (i.e. that you are not usingP PYCa) when contributing data to the program and when publishing elsewhere.

Link to this post | posted 06 Sep, 2024 23:42

viknesh

serivas
Hello all,

Could "Bacto" Tryptone (pancreatic digest of casein - microbiological peptone) be used to replace the "fisher" brand peptone we are currently using for our top agar?

It is likley that you'll be able to culture the bacteria we use for phage-hunting just as well using peptone or tryptone. However, for us to compare data from across the SEA program, I would recommend using peptone. If you use tryptone, it will be important for you to disclose this information (i.e. that you are not usingP PYCa) when contributing data to the program and when publishing elsewhere.

Posted in: Phage Discovery/Isolation → Precipitation of CaCl2 in PYCA top-agar

Link to this post \| posted 03 Sep, 2024 13:51
viknesh	engstrom Hi Folks, However, what Eric Engstrom should have written would be "we switched from Fisher Brand Peptone to Bacto Peptone." I should read my notes before posting anything. We did indeed try the agar switch first, but as I review this, that does not appear to have been the fix. The subsequent shift to Bacto Peptone is what saved our bacon. Heartfelt apologies for disseminating inaccurate information. I was traveling and relying on my memory, rather than my notebook. Never a good idea. Eric No apologies necessary, Eric. This is why this forum can be so valuable – thanks for so quickly responding to this post. We'll try to find additional ways to disseminate this info. Vic

Link to this post | posted 03 Sep, 2024 13:51

viknesh

engstrom
Hi Folks,

However, what Eric Engstrom should have written would be "we switched from Fisher Brand Peptone to Bacto Peptone." I should read my notes before posting anything. We did indeed try the agar switch first, but as I review this, that does not appear to have been the fix. The subsequent shift to Bacto Peptone is what saved our bacon.

Heartfelt apologies for disseminating inaccurate information. I was traveling and relying on my memory, rather than my notebook. Never a good idea.

Eric

No apologies necessary, Eric. This is why this forum can be so valuable – thanks for so quickly responding to this post. We'll try to find additional ways to disseminate this info.

Vic

Posted in: Phage Discovery/Isolation → Precipitation of CaCl2 in PYCA top-agar

Link to this post \| posted 03 Sep, 2024 11:46
viknesh	Hi all, Eric Engstrom recently shared the following: "Our problems finally went away when we switched from Fisher Brand Agar to Bacto Agar. With Bacto Agar, we did not need to drop the CaCl2 level and we could return to using our regular DI water. The stuff is expensive, so we have a container of Bacto Agar labeled "Top-agar Only" and we still use the Fisher Agar with no issue for our plate media.

Link to this post | posted 03 Sep, 2024 11:46

viknesh

Hi all,

Eric Engstrom recently shared the following:

"Our problems finally went away when we switched from Fisher Brand Agar to Bacto Agar. With Bacto Agar, we did not need to drop the CaCl2 level and we could return to using our regular DI water. The stuff is expensive, so we have a container of Bacto Agar labeled "Top-agar Only" and we still use the Fisher Agar with no issue for our plate media.

Posted in: Phage Discovery/Isolation → Precipitation of CaCl2 in PYCA top-agar

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