SEA-PHAGES | All posts created by Thiel

Link to this post \| posted 12 Jan, 2024 18:30
Thiel	Dear David, I am truly grateful for everything. The amazing impact you have had on both myself and HHMI is beyond measure! Best, Sarah Sarah J. Swerdlow Assistant Professor University of Pitt, Greensburg sjs308@pitt.edu

Posted in: General Message Board → A Message from David Asai

Link to this post \| posted 06 Apr, 2023 16:48
Thiel	Hi, I can share a photo but Kristen shared a great suggestion with me that worked. We put 150uL of phage buffer in a tube and then picked 15-20 plaques from the same plate. We had done 4 purifications so we were confident that it was pure. We put the 15-20 plaques in the 150uL of phage buffer. We then did serial dilutions, infected the bacteria and plated as normal the 10^0 through 10^-4 and the 10^-3 was very nicely webbed.

Link to this post | posted 06 Apr, 2023 16:48

Thiel

Hi, I can share a photo but Kristen shared a great suggestion with me that worked. We put 150uL of phage buffer in a tube and then picked 15-20 plaques from the same plate. We had done 4 purifications so we were confident that it was pure. We put the 15-20 plaques in the 150uL of phage buffer. We then did serial dilutions, infected the bacteria and plated as normal the 10^0 through 10^-4 and the 10^-3 was very nicely webbed.

Posted in: Phage Discovery/Isolation → Low plate titer issues

Link to this post \| posted 31 Mar, 2023 14:13
Thiel	I've had this same issue with a new turbid plaque, PhriedEgg. I'm not sure of an answer but if anyone has suggestions then that would be great. Thanks.

Posted in: Phage Discovery/Isolation → Low plate titer issues

Link to this post \| posted 31 Mar, 2023 14:10
Thiel	I've had this same issue with a new turbid plaque, PhriedEgg. I'm not sure of an answer but if anyone has suggestions then that would be great. Thanks.

Posted in: Phage Discovery/Isolation → Low plate titer issues

Link to this post \| posted 31 Aug, 2020 19:16
Thiel	Hello, I'm wondering if when using PECAAN we are only supposed to click on the evidence if it supports our function? For one of our NKF genes, HHPRED is giving us a DNA-binding domain with 38% coverage. I know that this is not good evidence for a function for our gene but it is evidence that we are going to call it NKF - so the question is do we check the box? NCBI blast has 100% identity and aligned with hypothetical protein so we would definitely check that box. The conserved domain database shows nothing and the Transmembrane prediction shows that it's not a transmembrane protein. Do we click the use as evidence box to show that we looked at it and we think that its is not a transmembrane protein? I hope that the question makes sense. Thanks, Sarah

Link to this post | posted 31 Aug, 2020 19:16

Thiel

Hello,
I'm wondering if when using PECAAN we are only supposed to click on the evidence if it supports our function? For one of our NKF genes, HHPRED is giving us a DNA-binding domain with 38% coverage. I know that this is not good evidence for a function for our gene but it is evidence that we are going to call it NKF - so the question is do we check the box?
NCBI blast has 100% identity and aligned with hypothetical protein so we would definitely check that box.
The conserved domain database shows nothing and the Transmembrane prediction shows that it's not a transmembrane protein. Do we click the use as evidence box to show that we looked at it and we think that its is not a transmembrane protein?

I hope that the question makes sense.

Thanks,
Sarah

Posted in: PECAAN → Clicking Supporting Evidence in PECAAN

Link to this post \| posted 21 Feb, 2020 20:56
Thiel	Great. Thank you!

Posted in: Phage Discovery/Isolation → Phages suddenly not fully lysing on M. foliorum

Link to this post \| posted 21 Feb, 2020 20:43
Thiel	Hello, I think that we are having a similar problem to the post above that says "D29 plaques barely visible on M. smeg" but I'm not sure. We are doing the phage isolation for the 2nd semester (so I'm not very experienced) and the students were on their 2nd round of purifications and starting to do the 3rd round and then make webbed plates. Our host is M. foliorum. I take new M. foliorum out of the -80C every 2 weeks, streak a plate and then shake it to make new stocks for them to use. The entire class started getting very similar looking plaques, that were difficult to see and smelled weird. (See image). The plates labeled "old" bacteria is what we had been using. The plates labeled "new" bacteria was when we picked a new colony from the same streak plate and it starting giving us the odd results. The difference in the incubation times was because we thought if we gave the 10^-3 plate a little more time- it would fully lyse, but it didn't. We picked a another new colony from the M. foliorum streak plate, grew it up and it seems to have fixed the problem (no pic). Just in case, I made a new M. foliorum streak plate from a different glycerol stock and will be growing that up for future use for the students. So we might have fixed the problem, but what do you think happened? Did we get lucky and pick a mutant colony? Any insight would be awesome. I'll let you know if the problem is not fixed. 1.27Mb

Link to this post | posted 21 Feb, 2020 20:43

Thiel

Hello,
I think that we are having a similar problem to the post above that says "D29 plaques barely visible on M. smeg" but I'm not sure.
We are doing the phage isolation for the 2nd semester (so I'm not very experienced) and the students were on their 2nd round of purifications and starting to do the 3rd round and then make webbed plates. Our host is M. foliorum. I take new M. foliorum out of the -80C every 2 weeks, streak a plate and then shake it to make new stocks for them to use. The entire class started getting very similar looking plaques, that were difficult to see and smelled weird. (See image). The plates labeled "old" bacteria is what we had been using. The plates labeled "new" bacteria was when we picked a new colony from the same streak plate and it starting giving us the odd results. The difference in the incubation times was because we thought if we gave the 10^-3 plate a little more time- it would fully lyse, but it didn't.

We picked a another new colony from the M. foliorum streak plate, grew it up and it seems to have fixed the problem (no pic). Just in case, I made a new M. foliorum streak plate from a different glycerol stock and will be growing that up for future use for the students.

So we might have fixed the problem, but what do you think happened? Did we get lucky and pick a mutant colony? Any insight would be awesome. I'll let you know if the problem is not fixed.

Posted in: Phage Discovery/Isolation → Phages suddenly not fully lysing on M. foliorum

Link to this post \| posted 03 Jun, 2019 18:29
Thiel	Hello, This was my first time running the phage discovery semester and some of the DNA was smeared. We used M. foliorum as our host. We used the nuclease mix from Sigma-aldrich (#GE80-6501-421018-03) and did the EDTA, proteinase K, and used the wizard kit. We tried it the first time and ran uncut and cut with RE. All of the gels/ phages except one, were smeared. Renzie was the best and still smeared some. We isolated DNA again and just ran uncut. From the uncut DNA gel image, Dan was able to sequence DNA from Renzie and Lenlyn. Thanks, Sarah 926Kb

Link to this post | posted 03 Jun, 2019 18:29

Thiel

Hello,
This was my first time running the phage discovery semester and some of the DNA was smeared. We used M. foliorum as our host. We used the nuclease mix from Sigma-aldrich (#GE80-6501-421018-03) and did the EDTA, proteinase K, and used the wizard kit. We tried it the first time and ran uncut and cut with RE. All of the gels/ phages except one, were smeared. Renzie was the best and still smeared some. We isolated DNA again and just ran uncut. From the uncut DNA gel image, Dan was able to sequence DNA from Renzie and Lenlyn.

Thanks,
Sarah

Posted in: Phage Biology → DNA Smear

Link to this post \| posted 22 Mar, 2019 18:36
Thiel	Hello, This is my first time running SEA-PHAGES and we have some 10^11 and 10^12 titers. I saw this post but I wasn't sure if we needed to dilute them first because I remember at a meeting someone mentioning that it needed to be diluted if the titer was really high. I can't find it in the protocol now but I remember it being talked about a lot. Suggestions? Just try it and see what happens or should I try diluting it? Thanks, Sarah

Link to this post | posted 22 Mar, 2019 18:36

Thiel

Hello,

This is my first time running SEA-PHAGES and we have some 10^11 and 10^12 titers. I saw this post but I wasn't sure if we needed to dilute them first because I remember at a meeting someone mentioning that it needed to be diluted if the titer was really high. I can't find it in the protocol now but I remember it being talked about a lot. Suggestions? Just try it and see what happens or should I try diluting it?

Thanks,
Sarah

Posted in: Phage Discovery/Isolation → new DNA protocol with really high titer lysates

Link to this post \| posted 21 Mar, 2019 14:08
Thiel	Debbie, This makes sense. Thank you! Sarah

Posted in: Host-Range Project → Basic Host Range Project Information

Recent Activity

All posts created by Thiel