SEA-PHAGES | All posts created by viknesh

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Link to this post \| posted 18 Nov, 2025 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 18 Nov, 2025 22:47

Link to this post | posted 18 Nov, 2025 22:46

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 18 Nov, 2025 22:47

Posted in: Phage Discovery/Isolation → 260/280 ratios too high from DNA extraction

Link to this post \| posted 18 Nov, 2025 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 18 Nov, 2025 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.

Posted in: Phage Discovery/Isolation → 260/280 ratios too high from DNA extraction

Link to this post \| posted 18 Nov, 2025 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 18 Nov, 2025 19:34

Link to this post | posted 18 Nov, 2025 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 18 Nov, 2025 19:34

Posted in: Phage Discovery/Isolation → 260/280 ratios too high from DNA extraction

Link to this post \| posted 14 Nov, 2025 19:43
viknesh	brueschhoff Hi Debbie, Thank you for your reply. I streaked for single colonies and then did three patch plates. Would it be advisable to streak more? Best, Beth Hi Beth, Reiterating Debbie's advice, please purify colonies at least twice before preparing a liquid culture. That means streaking a colonies once, then picking a colony from the new plate and doing another streak, and finally picking a new colony to be used to inoculate a liquid culture. If you want to perform a patch test, it should also be done using these colonies that have been purified at least twice. You'll want to do this for about 5 colonies. This is described in Protocol 11.2 of the new Guide. If those cultures dont saturate, then as Debbie said you might have unstable lysogens, which has been observed for other phages. But we can only draw that kind of conclusion if we can be certain that the colonies have been properly purified.

Link to this post | posted 14 Nov, 2025 19:43

viknesh

brueschhoff
Hi Debbie, Thank you for your reply. I streaked for single colonies and then did three patch plates. Would it be advisable to streak more?

Best,
Beth

Hi Beth,

Reiterating Debbie's advice, please purify colonies at least twice before preparing a liquid culture. That means streaking a colonies once, then picking a colony from the new plate and doing another streak, and finally picking a new colony to be used to inoculate a liquid culture. If you want to perform a patch test, it should also be done using these colonies that have been purified at least twice. You'll want to do this for about 5 colonies. This is described in Protocol 11.2 of the new Guide.

If those cultures dont saturate, then as Debbie said you might have unstable lysogens, which has been observed for other phages. But we can only draw that kind of conclusion if we can be certain that the colonies have been properly purified.

Posted in: Lysogeny/Immunity → Growing Liquid Cultures for lysogens

Link to this post \| posted 14 Nov, 2025 15:21
viknesh	alexander.baumann@tccd.edu Hello, After we prepare our grids, is there a suggested time in which to image them, and a maximum time we can store them before they're, on average, too degraded for TEM to visualize the bacteriophages? The grids should be stable for weeks to months, if not years. Once stained, you'll want to avoid exposure to moiture and direct light. You could put the grids (in their holder) in a small box with a dessicant.

Link to this post | posted 14 Nov, 2025 15:21

viknesh

alexander.baumann@tccd.edu
Hello,

After we prepare our grids, is there a suggested time in which to image them, and a maximum time we can store them before they're, on average, too degraded for TEM to visualize the bacteriophages?

The grids should be stable for weeks to months, if not years. Once stained, you'll want to avoid exposure to moiture and direct light. You could put the grids (in their holder) in a small box with a dessicant.

Posted in: Phage Biology → TEM

Link to this post \| posted 07 Nov, 2025 21:04
viknesh	The HHMI SEA Team is excited to share that we are looking to recruit a person to join us as a Program Officer. This position is posted on HHMI's career webpage and other websites. We hope that you will help share this opportunity with friends and colleagues in your network. The posting will remain up until November 21, and candidates must apply via the HHMI website. Thank you.

Posted in: General Message Board → New Program Officer Position with HHMI SEA

Link to this post \| posted 07 Nov, 2025 18:43
viknesh	pia1@pitt.edu Thanks, Debbie and Vik. I’ve tried several times to attach the results slide as a PPT or JPG, but it isn’t going through. Would it be okay if I email the results instead? I’m trying to share the superinfection immunity test outcomes for phages Dmitri and Arri. Dmitri behaved as expected (no clearing or plaques on the lysogen lawn), but Arri’s results were unexpected (EOP ~10⁻⁴). I’d appreciate any thoughts you might have. Of course, we’ll need to repeat the experiments. Ping, have you been able to reproduce these results? As an aside, when spotting a dilution series on a plate, I would avoid circling each spot (or at least erasing the circles before imaging) as they are distracting and not great for publication. Since we'd love to be able to use these data for potential publications, consider just listing the dilutions next to the spots or preparing a grid. Edited 07 Nov, 2025 18:49

Link to this post | posted 07 Nov, 2025 18:43

viknesh

pia1@pitt.edu
Thanks, Debbie and Vik. I’ve tried several times to attach the results slide as a PPT or JPG, but it isn’t going through. Would it be okay if I email the results instead? I’m trying to share the superinfection immunity test outcomes for phages Dmitri and Arri. Dmitri behaved as expected (no clearing or plaques on the lysogen lawn), but Arri’s results were unexpected (EOP ~10⁻⁴). I’d appreciate any thoughts you might have. Of course, we’ll need to repeat the experiments.

Ping, have you been able to reproduce these results?

As an aside, when spotting a dilution series on a plate, I would avoid circling each spot (or at least erasing the circles before imaging) as they are distracting and not great for publication. Since we'd love to be able to use these data for potential publications, consider just listing the dilutions next to the spots or preparing a grid.

Edited 07 Nov, 2025 18:49

Posted in: Lysogeny/Immunity → Superinfection immunity

Link to this post \| posted 07 Nov, 2025 15:54
viknesh	pia1@pitt.edu Does superinfection immunity completely block a lysogen from being reinfected by the same temperate phage that established the lysogeny? In general, homotypic superinfection immunity is complete, meaning that a given phage will not be able to complete a productive infection in a lysogen of that phage – you wont expect to see plaques. At some low frequency, you can expect to see plaques. These are often the result of a mutant phage within the population of phage in your lysate that cannot be repressed. You might imagine, for example, a mutation in the binding site of the immunity repressor that weakens binding and repression. If your phage is behaving counter to these generalized outcomes, please do share!

Link to this post | posted 07 Nov, 2025 15:54

viknesh

pia1@pitt.edu
Does superinfection immunity completely block a lysogen from being reinfected by the same temperate phage that established the lysogeny?

In general, homotypic superinfection immunity is complete, meaning that a given phage will not be able to complete a productive infection in a lysogen of that phage – you wont expect to see plaques. At some low frequency, you can expect to see plaques. These are often the result of a mutant phage within the population of phage in your lysate that cannot be repressed. You might imagine, for example, a mutation in the binding site of the immunity repressor that weakens binding and repression. If your phage is behaving counter to these generalized outcomes, please do share!

Posted in: Lysogeny/Immunity → Superinfection immunity

Link to this post \| posted 07 Nov, 2025 14:48
viknesh	liltye01 Good morning All, Would someone be able to point me in the right direction to find the TEM information from HHMI. I remember when we were in Maryland we discussed an option to send our samples back there for TEM and we are at that point now, so I need the contact information, mailing address and procedures for packing. Thanks You are referring to the Imaging Facilty run by UMBC, which images phages for many schools running SEA-PHAGES but it is otherwise not affiliated with HHMI. Their phage imaging services (pricing etc.) can be found on their website, here: https://kpif.umbc.edu/bacteriophage-imaging/

Link to this post | posted 07 Nov, 2025 14:48

viknesh

liltye01
Good morning All,
Would someone be able to point me in the right direction to find the TEM information from HHMI. I remember when we were in Maryland we discussed an option to send our samples back there for TEM and we are at that point now, so I need the contact information, mailing address and procedures for packing.

Thanks

You are referring to the Imaging Facilty run by UMBC, which images phages for many schools running SEA-PHAGES but it is otherwise not affiliated with HHMI. Their phage imaging services (pricing etc.) can be found on their website, here: https://kpif.umbc.edu/bacteriophage-imaging/

Posted in: Phage Biology → TEM

Link to this post \| posted 15 Oct, 2025 17:03
viknesh	jayadasgupta Hello everyone, I have been getting broth cultures ready from agar plates (isolated colonies). I was wondering if it is ok to subculture the broth Arthrobacter for next day's experiments? Meaning, take 1ml of the overnight broth and add to 50ml fresh broth for next day's lab? Or is that a no-no? And that we should start from the plate every time?. Thank you, Jaya. The recommendation is to minimize the number of passages, as each passage increases the genetic variation in the population and possibly phenotypes important for infection. For a reliable culture, we recommend always starting a culture from a colonies off a freshly streaked plate. In a pinch, you can subculture from an existing culture, but I would do this regularly.

Link to this post | posted 15 Oct, 2025 17:03

viknesh

jayadasgupta
Hello everyone,
I have been getting broth cultures ready from agar plates (isolated colonies). I was wondering if it is ok to subculture the broth Arthrobacter for next day's experiments? Meaning, take 1ml of the overnight broth and add to 50ml fresh broth for next day's lab? Or is that a no-no? And that we should start from the plate every time?.

Thank you,
Jaya.

The recommendation is to minimize the number of passages, as each passage increases the genetic variation in the population and possibly phenotypes important for infection. For a reliable culture, we recommend always starting a culture from a colonies off a freshly streaked plate. In a pinch, you can subculture from an existing culture, but I would do this regularly.

Posted in: Phage Discovery/Isolation → Sub-culturing from PyCa Broth

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