Link to this post | posted 29 Sep, 2023 03:47

uOttawaPHAGE

Hi Kyle. We think we are having an issue similar to what you describe above. We have just tried new agar and it seems to have helped but not completely. Given the contaminant survives the autoclave, it could be in some of the bottles that are filled with top agar, so I suppose it could persist. I’m hopeful this will fix the problem. Two questions: 1. In your thread you mention you were also having issues with phage propagation, which it sounded like you linked to the top agar issue. Did the propagation issue resolve when the top agar issue resolved? I’m assuming yes, but you didn't say it above. We are seeing propagation issues, but only for a subset of phages. The phages were found on new hosts for us, A. glob 2979 and A. sulf, and we aren't sure if this issue could be more common for these hosts, or if it is linked to the top agar. 2. We aren't seeing contamination in our plates, which is confusing to me. We wonder if this could be because of the cycloheximide. Did you ever consider this? This would suggest a fungal contaminant. Adam

Link to this post | posted 14 Jul, 2023 06:44

uOttawaPHAGE

mdgainey Can anyone advise on whether the dry uranyl acetate waste generated from the EM staining process is considered exempt waste because of the very small dry amounts of waste, or if it has to be disposed of via a radioactive waste route? I am getting ready to write an SOP for staining using uranyl acetate at my institution and want to make sure that I handle the waste properly/prepare for the costs ect. hi Maria. I have inquired at my institution and uranyl acetate seems to exist in a nether state between radioactive and chemical waste, and neither of the individuals responsible for these two wastes want to claim it. Hopefully it will be more straightforward at your institution.

Link to this post | posted 11 Apr, 2023 19:17

uOttawaPHAGE

Hi Michèle. I quickly looked at the HHpred results for these two genes. IF you look down your list their are hits with HTH domains, and many of your hits are either sigma factors or related to sigma factors, which have HTH domains. Upon closer inspection the matches all appear to be minimal domains that have three (in one or two cases only two) alpha helical domains with short non-helical segments. I believe they both meet the threshold of having a HTH domain. I still struggle with what this function should be. I think "HTH domain" or ""HTH domain containing protein" would be best, but I don't thinks these are options on the approved function list. Many of the hits are to transcription factors that have HTH domains, so they could be "HTH DNA binding domain protein" but it does seem like there isn't sufficient data to call these "DNA binding proteins." Debbie will need to weigh in on this. It would seem like we need a new more minimal function for these types of proteins. Adam

Link to this post | posted 28 Mar, 2023 20:04

uOttawaPHAGE

thanks Debbie. We will leave it NKF and I'll suggest the students see what they can learn from published work or if any of the foldseek matches are cryoEMs from a podo. I also suggested they see if some of the other T4 proteins (gp11?) is present in the genome. I was mainly struck by the alphafold/HHpred similarity, but it sounds like there is currently insufficient data to give it a specific call. Have you gotten similar questions from all annotators of this pham? Adam

Link to this post | posted 24 Mar, 2023 20:18

uOttawaPHAGE

Kikiko_42 (46820-49244) is in a pham that has been called NKF in all other annotations, but it has very strong HHpred hits to gp12 in T7 (and other phages) that is part of the DNA ejection machinery. On Alphafold it also has very high probability to have a similar structure to gp12. I'm trying to understand why it has no approved function despite - is it because unlike T7, which has a contractile tail, this gene is found in podoviridaes? Without experimental data do we leave it as an NKF? Given that this pham has many members, I assume you have answered this question before…. Adam

Link to this post | posted 03 Feb, 2023 19:13

uOttawaPHAGE

I have a similar question about Polkaroo, a P1 cluster phage. Polkaroo_12 (7912-8325) (current pham# 68611) is annotated as NKF for virtually every pham member, but has high HHpred hits to a putative tail component and a minor capsid protein. Similar hits to HK97_10, but also other proteins. See: https://www.ebi.ac.uk/interpro/entry/InterPro/IPR021080/ Why are so many of the hits calling it a minor capsid protein?

Link to this post | posted 02 Nov, 2022 20:30

uOttawaPHAGE

Hi Dan. We're finally about to do our inaugural MiSeq run with ~20 libraries. A few final questions, if you have time: 1. We were going to use the Illumina Protocol A for denaturing/loading - I'm assuming using Bioanalyzer results constitutes "standard normalization"? 2. Do you denature 5uL of 4nM pooled libraries? 3. And to clarify, with 20 libraries each at 4nM, that would only be 0.25uL of each library. 4. IN a response above you mentioned your libraries are often not much above 4nM. Ours are higher, so I'm wondering if we did our library prep differently. We used the NEBNext Ultra II FS kit. How much input genome to you use? 5. Do you add 1% PhiX as a control? This seems standard. 6. Any other subtleties I should know about? thanks again for your help with these questions. Adam

Link to this post | posted 01 Aug, 2022 12:58

uOttawaPHAGE

Hi Dan. We are finally making our libraries and I was curious if you use the Bioanalyzer to check the library - based on the question above from Fernando it sounds like you do. Some questions: 1. I've not worked with data off the bioanalyzer - is there a cut-off for how you determine if a library is good enough to sequence? 2. Do you use the Qubit before or after library prep? for some reason I thought you used it after, but I'm thinking I have that wrong and you use it before to assess the quality/quantity of the genome. thanks again for your help with these questions. Adam

Link to this post | posted 01 Aug, 2022 12:55

uOttawaPHAGE

Hi Dan. We are finally making our libraries and I was curious if you use the Bioanalyzer to check the library - based on the question above from Fernando it sounds like you do. Some questions: 1. I've not worked with data off the bioanalyzer - is there a cut-off for how you determine if a library is good enough to sequence? 2. Do you use the Qubit before or after library prep? for some reason I thought you used it after, but I'm thinking I have that wrong and you use it before to assess the quality/quantity of the genome. thanks again for your help with these questions. Adam

Link to this post | posted 10 Apr, 2022 13:19
uOttawaPHAGE	yes, 17167. I was referring to the G at the start of the slippery sequence. We will call this frameshift for both Ottawa and Kharcho. The 54457 Pham has 22 members. 2 have called the frameshift (phiSASD1 and Dubu), the rest have not. Should we investigate and make those calls too? Also, any opinion about the very small extension? Is homology to lambda unsual for Actino TACs?