SEA-PHAGES | All posts created by nic.vega

Link to this post \| posted 20 Dec, 2024 02:56
nic.vega	Vic - I think you're right. We set up (with TrixiePhattel again) standard 1h digests, then a microwave digest and a "standard" 37C digest starting and ending at the same time as the microwave - with handling time, it turned out to be 2 minutes. Overall the banding patterns are very similar except for maybe some differences in undigested band weight, even for PstI which should only have 50% activity in CutSmart. And that's with intentionally overloading the reactions - should be >1 ug DNA/rxn - to really give the enzymes something to do. 134Kb

Link to this post | posted 20 Dec, 2024 02:56

Vic - I think you're right. We set up (with TrixiePhattel again) standard 1h digests, then a microwave digest and a "standard" 37C digest starting and ending at the same time as the microwave - with handling time, it turned out to be 2 minutes. Overall the banding patterns are very similar except for maybe some differences in undigested band weight, even for PstI which should only have 50% activity in CutSmart. And that's with intentionally overloading the reactions - should be >1 ug DNA/rxn - to really give the enzymes something to do.

Posted in: Phage Discovery/Isolation → did you know you can do restriction digests in the microwave?

Link to this post \| posted 10 Dec, 2024 18:11
nic.vega	We did some side-by-side reactions on TrixiePhattel; results attached. The gel was much better this time - it turns out the reason that the first gel was blurry is that the 10X TBE in the teaching lab expired in 2012. 154Kb

Posted in: Phage Discovery/Isolation → did you know you can do restriction digests in the microwave?

Link to this post \| posted 05 Dec, 2024 22:22
nic.vega	Sure, we can give it a try! I'll plan on having some of my folks try microwave vs traditional digests with some commonly-used enzymes and see how they compare.

Posted in: Phage Discovery/Isolation → did you know you can do restriction digests in the microwave?

Link to this post \| posted 05 Dec, 2024 20:22
nic.vega	really! Found here, protocol is: Make reactions as usual, in 1.5 mL tubes (0.2 mL PCR tubes can melt) Arrange tubes so they are off center on the microwave platform Run four cycles of 10 seconds on, 10 seconds off Add loading dye, load & run gel Be amazed Gel image is a HindIII digest of TrixiePhattel genome (with too much DNA per lane, so kind of smear-y). Numbers above lanes are the number of on/off cycles. Reference: Das RH, Ahirwar R, Kumar S, Nahar P. Microwave-mediated enzymatic modifications of DNA. Anal Biochem. 2015 Feb 15;471:26-8. doi: 10.1016/j.ab.2014.11.003. Epub 2014 Nov 13. PMID: 25447491. 88Kb

Link to this post | posted 05 Dec, 2024 20:22

nic.vega

really! Found here, protocol is:

Make reactions as usual, in 1.5 mL tubes (0.2 mL PCR tubes can melt)

Arrange tubes so they are off center on the microwave platform

Run four cycles of 10 seconds on, 10 seconds off

Add loading dye, load & run gel

Be amazed
Gel image is a HindIII digest of TrixiePhattel genome (with too much DNA per lane, so kind of smear-y). Numbers above lanes are the number of on/off cycles.

Reference: Das RH, Ahirwar R, Kumar S, Nahar P. Microwave-mediated enzymatic modifications of DNA. Anal Biochem. 2015 Feb 15;471:26-8. doi: 10.1016/j.ab.2014.11.003. Epub 2014 Nov 13. PMID: 25447491.

Posted in: Phage Discovery/Isolation → did you know you can do restriction digests in the microwave?

Link to this post \| posted 15 Nov, 2024 19:24
nic.vega	We're having a go at library prep, but the core here apparently isn't sure where to start for non-mammalian DNA. Does anyone have settings for Covaris (E220) (we're sequencing on iSeq 2x150, shooting for fragments ~400 bp)? From restriction gels, I think our genomes are all around average size for globi phage (~40-50 kb).

Posted in: Sequencing, Assembling, and Finishing Genomes → Covaris fragmentation

Link to this post \| posted 26 Oct, 2024 14:52
nic.vega	If your students are still struggling with webbed plates, particularly for low-yield or small plaque phage, we've had good luck (1) increasing the incubation time of bacteria + phage before making plaque plates to allow a lysis cycle (I'm using 1h instead of 15 minutes with globi as host, your timing may vary) and (2) dropping the top agar concentration in PYCa to 0.2-0.3% by diluting with phage buffer - this also provides 2.5-5% glycerol, which can help increase plaque size (Santos, Sílvio B., Carla M. Carvalho, Sanna Sillankorva, Ana Nicolau, Eugénio C. Ferreira, and Joana Azeredo. 2009. “The Use of Antibiotics to Improve Phage Detection and Enumeration by the Double-Layer Agar Technique.” BMC Microbiology 9 (1): 148. https://doi.org/10.1186/1471-2180-9-148.) 73Kb

Link to this post | posted 26 Oct, 2024 14:52

nic.vega

If your students are still struggling with webbed plates, particularly for low-yield or small plaque phage, we've had good luck (1) increasing the incubation time of bacteria + phage before making plaque plates to allow a lysis cycle (I'm using 1h instead of 15 minutes with globi as host, your timing may vary) and (2) dropping the top agar concentration in PYCa to 0.2-0.3% *by diluting with phage buffer* - this also provides 2.5-5% glycerol, which can help increase plaque size (Santos, Sílvio B., Carla M. Carvalho, Sanna Sillankorva, Ana Nicolau, Eugénio C. Ferreira, and Joana Azeredo. 2009. “The Use of Antibiotics to Improve Phage Detection and Enumeration by the Double-Layer Agar Technique.” BMC Microbiology 9 (1): 148. https://doi.org/10.1186/1471-2180-9-148.)

Posted in: Phage Discovery/Isolation → Webbed plate hack for low-titer stocks and small plaques

Link to this post \| posted 26 Oct, 2024 14:35
nic.vega	A few newer papers (and one old one) that may be of interest: Gallet, Romain, Sherin Kannoly, and Ing-Nang Wang. 2011. “Effects of Bacteriophage Traits on Plaque Formation.” BMC Microbiology 11 (1): 181. https://doi.org/10.1186/1471-2180-11-181. Germida, James J., and L. E. Casida. 1981. “Isolation of Arthrobacter Bacteriophage from Soil.” Applied and Environmental Microbiology 41 (6): 1389–93. Keen, Eric C. 2015. “A Century of Phage Research: Bacteriophages and the Shaping of Modern Biology.” BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology 37 (1): 6–9. https://doi.org/10.1002/bies.201400152. Zrelovs, Nikita, Andris Dislers, and Andris Kazaks. 2021. “Motley Crew: Overview of the Currently Available Phage Diversity.” Frontiers in Microbiology 11 (January). https://doi.org/10.3389/fmicb.2020.579452.

Link to this post | posted 26 Oct, 2024 14:35

nic.vega

A few newer papers (and one old one) that may be of interest:

Gallet, Romain, Sherin Kannoly, and Ing-Nang Wang. 2011. “Effects of Bacteriophage Traits on Plaque Formation.” BMC Microbiology 11 (1): 181. https://doi.org/10.1186/1471-2180-11-181.

Germida, James J., and L. E. Casida. 1981. “Isolation of Arthrobacter Bacteriophage from Soil.” Applied and Environmental Microbiology 41 (6): 1389–93.

Keen, Eric C. 2015. “A Century of Phage Research: Bacteriophages and the Shaping of Modern Biology.” BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology 37 (1): 6–9. https://doi.org/10.1002/bies.201400152.

Zrelovs, Nikita, Andris Dislers, and Andris Kazaks. 2021. “Motley Crew: Overview of the Currently Available Phage Diversity.” Frontiers in Microbiology 11 (January). https://doi.org/10.3389/fmicb.2020.579452.

Posted in: Papers → Introductory Phage Papers

Link to this post \| posted 11 Oct, 2024 13:40
nic.vega	Did you change batches of media around the time plating started to fail? In particular, if the Mg/Ca levels are off in the PYCa, that can significantly affect phage sorption & plating efficiency.

Posted in: Arthrobacter → Arthrobacter vanishing plaques

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