SEA-PHAGES | A atrocyaneus DNA extraction

Link to this post \| posted 30 Mar, 2024 20:23
tperezmo	Hi all, I recently switched one section to work on A. atrocyaneus while the others work on A. globiformis. For A. globiformis, we always have really good DNA yields 40 - 100 ng per ul. However, for A. atrocyaneus, we are not hitting even 20 ng / ul. I have used the original protocol with nucleases, SDS, EDTA, Proteinase K. The results are for all students in the class, for 2 semesters now. When trying to do the restriction digests, we can barely see a band in the controls. I have looked at the Microbacterium forum but there it seems it's a lysate concentrations. All my students have at least 1 x 10^8 or more. Any thoughts? Thank you!

Link to this post | posted 30 Mar, 2024 20:23

Hi all,

I recently switched one section to work on A. atrocyaneus while the others work on A. globiformis. For A. globiformis, we always have really good DNA yields 40 - 100 ng per ul. However, for A. atrocyaneus, we are not hitting even 20 ng / ul. I have used the original protocol with nucleases, SDS, EDTA, Proteinase K. The results are for all students in the class, for 2 semesters now.
When trying to do the restriction digests, we can barely see a band in the controls.
I have looked at the Microbacterium forum but there it seems it's a lysate concentrations. All my students have at least 1 x 10^8 or more.
Any thoughts? Thank you!

Link to this post \| posted 31 Mar, 2024 02:59
viknesh	tperezmo Hi all, I recently switched one section to work on A. atrocyaneus while the others work on A. globiformis. For A. globiformis, we always have really good DNA yields 40 - 100 ng per ul. However, for A. atrocyaneus, we are not hitting even 20 ng / ul. I have used the original protocol with nucleases, SDS, EDTA, Proteinase K. The results are for all students in the class, for 2 semesters now. When trying to do the restriction digests, we can barely see a band in the controls. I have looked at the Microbacterium forum but there it seems it's a lysate concentrations. All my students have at least 1 x 10^8 or more. Any thoughts? Thank you! Hi Tiara, I don't have experience working with A. atrocyaneus, but a couple of things. 1. Have you considered using ZnCl2 precicptation? It seems to do the trick for folks with low yield. Also, if your titers are generally around 10^8, you might want to see if your titer is the issue. Do you still get low DNA yield from a lysate with a titer of 5 x 10^9 pfu/ml? 2. A quick search on phagesDB shows that Pitt and UCLA have submitted DNA for a few phages isolated on this host last year. I'd recommend pinging SEA faculty Kristen Butela and Amanda Friese for input (and have the respond on the forum so we capture it here for others too). Thanks. Vic

Link to this post | posted 31 Mar, 2024 02:59

viknesh

tperezmo
Hi all,

I recently switched one section to work on A. atrocyaneus while the others work on A. globiformis. For A. globiformis, we always have really good DNA yields 40 - 100 ng per ul. However, for A. atrocyaneus, we are not hitting even 20 ng / ul. I have used the original protocol with nucleases, SDS, EDTA, Proteinase K. The results are for all students in the class, for 2 semesters now.
When trying to do the restriction digests, we can barely see a band in the controls.
I have looked at the Microbacterium forum but there it seems it's a lysate concentrations. All my students have at least 1 x 10^8 or more.
Any thoughts? Thank you!

Hi Tiara,

I don't have experience working with A. atrocyaneus, but a couple of things.

1. Have you considered using ZnCl2 precicptation? It seems to do the trick for folks with low yield. Also, if your titers are generally around 10^8, you might want to see if your titer is the issue. Do you still get low DNA yield from a lysate with a titer of 5 x 10^9 pfu/ml?

2. A quick search on phagesDB shows that Pitt and UCLA have submitted DNA for a few phages isolated on this host last year. I'd recommend pinging SEA faculty Kristen Butela and Amanda Friese for input (and have the respond on the forum so we capture it here for others too).

Thanks.
Vic

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A atrocyaneus DNA extraction