SEA-PHAGES | All posts created by cellokiwi

Link to this post \| posted 15 May, 2024 16:19
cellokiwi	Hi Claire, I am having an issue where Aragorn is calling A BUNCH of tRNAs when I manually enter the FASTA and PECAAN is calling none of them. I went to put them in with the "Add a gene" button but it only lets me put in stop coordinates, not start. What am I missing? Thanks!

Posted in: PECAAN → New Features in PECAAN

Link to this post \| posted 11 Jul, 2023 17:59
cellokiwi	Hi Dan, I tried to do this and got the error attached in the screenshot. How do I fix this as DNA Master keeps giving me an "FTP Failure" error when I try to update or close it. DanRussell Hi Kathleen, Looks like that screenshot shows someone using a Windows VM within a Mac, so they can't use Chris or Debbie's procedures, which would be Mac-specific. Instead, they can follow the directions here to connect to an FTP site from Windows: https://www.howtogeek.com/272176/how-to-connect-to-ftp-servers-in-windows-without-extra-software/ Basically: 1. In Windows, open up a File Explorer window either using the Start Menu or clicking the little folder icon at the bottom of the screen. 2. In that File Explorer window, click on "This PC" on the left. 3. Right-click in the blank area called "Drives and Devices", and select "Add a network location" from the dropdown menu. 4. Then click "Choose a custom network location". 5. When it asks for an internet address, put "ftp://cobamide2.bio.pitt.edu/", then on the next screen make sure "Log in anonymously" is checked. 6. Complete the wizard with the open now option selected. 7. You should now see a folder open with the contents of that FTP site. Open the "DNAMas" folder, find the file "dna master.exe", and drag it to your desktop. Then you should be able to install! Much more complicated than it used to be, but hopefully this will work. –Dan 399Kb

Link to this post | posted 11 Jul, 2023 17:59

cellokiwi

Hi Dan,

I tried to do this and got the error attached in the screenshot. How do I fix this as DNA Master keeps giving me an "FTP Failure" error when I try to update or close it.

DanRussell
Hi Kathleen,

Looks like that screenshot shows someone using a Windows VM within a Mac, so they can't use Chris or Debbie's procedures, which would be Mac-specific. Instead, they can follow the directions here to connect to an FTP site from Windows:
https://www.howtogeek.com/272176/how-to-connect-to-ftp-servers-in-windows-without-extra-software/

Basically:
1. In Windows, open up a File Explorer window either using the Start Menu or clicking the little folder icon at the bottom of the screen.
2. In that File Explorer window, click on "This PC" on the left.
3. Right-click in the blank area called "Drives and Devices", and select "Add a network location" from the dropdown menu.
4. Then click "Choose a custom network location".
5. When it asks for an internet address, put "ftp://cobamide2.bio.pitt.edu/", then on the next screen make sure "Log in anonymously" is checked.
6. Complete the wizard with the open now option selected.
7. You should now see a folder open with the contents of that FTP site. Open the "DNAMas" folder, find the file "dna master.exe", and drag it to your desktop.

Then you should be able to install! Much more complicated than it used to be, but hopefully this will work.

–Dan

Posted in: DNA Master → DNA master server down?

Link to this post \| posted 17 Sep, 2022 19:15
cellokiwi	What pH is perfect for these bugs?

Posted in: Arthrobacter → Media pH

Link to this post \| posted 13 Sep, 2022 12:52
cellokiwi	So when you pass it, it goes back to yellow for a bit then turns orange again. We sadly don't have the resources to check 16S. Is there a resource for this through the program? The first time we saw it was in a lysogen so I just attributed it to wherever the genome had integrated itself messing with the color. Now, there has been zero change whatsoever. Same media, same CaCl2, same buffer, same incubator, same everything. One colleague suggested it is a carotenoid response to oxidative stress? Does that sound possible? Since we're using the same everything what would suddenly be stressing them out that wasn't before? In the picture the one on the left is a normal Arthrobacter culture and the one on the right is just starting to turn orange. They get darker than that. 107Kb

Link to this post | posted 13 Sep, 2022 12:52

cellokiwi

So when you pass it, it goes back to yellow for a bit then turns orange again. We sadly don't have the resources to check 16S. Is there a resource for this through the program? The first time we saw it was in a lysogen so I just attributed it to wherever the genome had integrated itself messing with the color. Now, there has been zero change whatsoever. Same media, same CaCl2, same buffer, same incubator, same everything. One colleague suggested it is a carotenoid response to oxidative stress? Does that sound possible? Since we're using the same everything what would suddenly be stressing them out that wasn't before?

In the picture the one on the left is a normal Arthrobacter culture and the one on the right is just starting to turn orange. They get darker than that.

Posted in: Arthrobacter → Orange Cells

Link to this post \| posted 12 Sep, 2022 16:19
cellokiwi	Has anyone ever had experience with Arthrobacter turning orange rather than yellowish? We are pretty sure there is no contamination, but the culture is orange suddenly.

Posted in: Arthrobacter → Orange Cells

Link to this post \| posted 31 Mar, 2022 14:55
cellokiwi	Debbie, Thank you SO much for the info. I feel much better for not being able to locate it since it is apparently a lot more complicated. Especially since it is a serine integrase.

Posted in: Lysogeny/Immunity → attP Location

Link to this post \| posted 29 Mar, 2022 20:01
cellokiwi	Hello, I'm trying to find the attP of my temperate phage. I know it will be directly downstream of the integrase gene. However, how do we find the exact bp number? Is there a tool that will search for it? Is there a particular consensus I should be looking for? Thanks!

Posted in: Lysogeny/Immunity → attP Location

Link to this post \| posted 06 Oct, 2021 23:45
cellokiwi	Hi, I was wondering if anyone knows of a good program/tool to construct circular diagrams of our genomes (given that they circularize almost immediately inside the host)? I'm wanting to relate gene expression data to location on the chromosome. Thanks in advance for your help!

Posted in: Bioinformatic Tools and Analyses → Circular Genome Visualization

Link to this post \| posted 13 Jan, 2020 21:01
cellokiwi	Hello Hive Mind I'm hoping I can get some help with a one step growth curve. My students have tried two different protocols, one of which works successfully in another mycobacteriophage lab, and we have not been able to get a nice S-curve. There is a trend that we see every time, high titer initially that continues to drop. The earliest time point we have tried (other than 0 min which has no plaques) is 10 min and I honestly refuse to believe that they are completing a replication cycle that quickly… So why do we have essentially inverse data to what you would expect? Again, this is highly repeatable; we have tried many times. I'm attaching a representative graph of what we are seeing. Thanks for any and all help we can get! 8Kb

Link to this post | posted 13 Jan, 2020 21:01

cellokiwi

Hello Hive Mind

I'm hoping I can get some help with a one step growth curve. My students have tried two different protocols, one of which works successfully in another mycobacteriophage lab, and we have not been able to get a nice S-curve. There is a trend that we see every time, high titer initially that continues to drop. The earliest time point we have tried (other than 0 min which has no plaques) is 10 min and I honestly refuse to believe that they are completing a replication cycle that quickly… So why do we have essentially inverse data to what you would expect? Again, this is highly repeatable; we have tried many times. I'm attaching a representative graph of what we are seeing. Thanks for any and all help we can get!

Posted in: Phage Biology → One Step Growth Curve

Link to this post \| posted 15 Oct, 2019 15:00
cellokiwi	We finally had success again by doing 2 things. We let the culture get older in the shaking flask before plating and we doubled the amount of bacteria (from 250 uL to 500uL) when we plated. I still don't know what to do about the fridge plates degrading. Letting them sit at room temp just makes this microbiologist get a nervous tick… lol. Good luck! UNHM Hello, We are having the exact same problem right now as you describe cellokiwi with A. globiformis. After incredible success in the first few several weeks with several of our students reaching high titer and others just needing to titer their likely high titer lysates, we have been met with a standstill for the last week and half unable to get an adequate lawn plate from our bacterial liquid culture(s). Most of our attempts at lawn plates have either given us NO lawn at all (after 24 hrs, our typical incubation at 30 degrees) or a very inconsistent/spotty lawn. We have restreaked from several glycerol stocks onto new PyCa plates and remade our liquid PyCa media and top agar. Any other thoughts would help as well for us in order to get our last students through the last steps and to the DNA extraction phase. Thank you, Kristen

Link to this post | posted 15 Oct, 2019 15:00

cellokiwi

We finally had success again by doing 2 things. We let the culture get older in the shaking flask before plating and we doubled the amount of bacteria (from 250 uL to 500uL) when we plated. I still don't know what to do about the fridge plates degrading. Letting them sit at room temp just makes this microbiologist get a nervous tick… lol. Good luck!

UNHM
Hello,
We are having the exact same problem right now as you describe cellokiwi with A. globiformis.
After incredible success in the first few several weeks with several of our students reaching high titer and others just needing to titer their likely high titer lysates, we have been met with a standstill for the last week and half unable to get an adequate lawn plate from our bacterial liquid culture(s).
Most of our attempts at lawn plates have either given us NO lawn at all (after 24 hrs, our typical incubation at 30 degrees) or a very inconsistent/spotty lawn.
We have restreaked from several glycerol stocks onto new PyCa plates and remade our liquid PyCa media and top agar.
Any other thoughts would help as well for us in order to get our last students through the last steps and to the DNA extraction phase.
Thank you,
Kristen

Posted in: Arthrobacter → Success followed by failure?

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