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Recent Activity
Low plate titer issues
Link to this post | posted 11 Apr, 2022 21:29 | |
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I have found and isolated a phage that I named Ascela. It hasn’t been confirmed yet, but I’m fairly certain that Ascela is a temperate phage. I have been trying to get Ascela to a higher titer, but I can’t seem to get good plated titers. The current titer that I calculated was 7.0x10^6. As I looked back through pictures of my full plate titers, I noticed that there are three plates present in every dilution that have a rapid decline in plaques present. I will go from a web plate and then suddenly have a significant drop in plaques (normally occurs in the 10^-1 to 10^-4 plates). This has been a reoccurring issue from the very beginning of isolating my phage and has been replicated in multiple different series of plates. I’ve made sure that I am vortexing them well before I plate them, but it’s not helping. I recently did another full plate titer, and it did the same thing it normally does. I wiped out the first two plates then had a web plate on 10^-2, but then on 10^-4 I only had sixteen plaques. There was nothing on 10^-5, 10^-6, or 10^-7, but then suddenly there was one plaque on the 10^-8 plate. I also did a DNA extraction following the SEAPhages protocol and got 226.8 ng/uL with 1.88 purity. So, I'm pretty sure that there is a fairly good titer in my lysate, but how do I get Ascela to titer well on a plate? Has anyone else had this issue where their plaques significantly drop after a web plate? If so, did you figure out how to fix it? Is there a different protocol that I can try to get a better full plate titer? Thank you in advance for your help. |
Link to this post | posted 31 Mar, 2023 14:10 | |
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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. |
Link to this post | posted 31 Mar, 2023 14:13 | |
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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. |
Link to this post | posted 31 Mar, 2023 14:37 | |
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Thiel Hi Sarah, Would you be able to share photos of what you are seeing? Vic |
Link to this post | posted 06 Apr, 2023 16:48 | |
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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 17:27 | |
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Thiel Great! |