SEA-PHAGES | All posts created by GregFrederick@letu.edu

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Link to this post \| posted 13 Jan, 2016 21:22
GregFrederick@letu.edu	Tamarah Adair We are running DNA Master within the WINE configuration. The installer has been copied and you should be able to get to it from this link. https://baylor.box.com/s/295to06pr1cpztqiu2jn0h77iy3vkanz Our contact is in Student Technology Services. He installs the DNA Master on the Macs in the lab at the beginning of the spring semester. If there are updates he incorporates these. I'm sure he would be glad to answer any questions you might have. Hey Tammy. I'm not a Mac guy… I used one some 20 years ago… but it has been a while. Your link above still works. It leads to file called DNAMaster_5.22.19.dmg Is that an executable file on Mac OS10? Does it need to be extracted? Or opened in another program? Sorry for the newb questions! Thanks! Greg

Link to this post | posted 13 Jan, 2016 21:22

Tamarah Adair
We are running DNA Master within the WINE configuration.
The installer has been copied and you should be able to get to it from this link.
https://baylor.box.com/s/295to06pr1cpztqiu2jn0h77iy3vkanz

Our contact is in Student Technology Services. He installs the DNA Master on the Macs in the lab at the beginning of the spring semester. If there are updates he incorporates these. I'm sure he would be glad to answer any questions you might have.

Hey Tammy. I'm not a Mac guy… I used one some 20 years ago… but it has been a while.

Your link above still works. It leads to file called DNAMaster_5.22.19.dmg

Is that an executable file on Mac OS10? Does it need to be extracted? Or opened in another program? Sorry for the newb questions!

Thanks! Greg

Posted in: DNA Master → Running DNA Master on a Mac using Wine

Link to this post \| posted 13 Jan, 2016 19:35
GregFrederick@letu.edu	Dan Russell Sharon Isern Can students get access to the VM 2016 via seaphages.org? Or do they have to be logged in as faculty? We're in class right now. Thanks!! Because of the software included, we couldn't post it totally publicly. And since there's no "registered student" accounts on PhagesDB, there's no way to give them access to the protected page. That said, I'll email you (and any other faculty who want it) the magic link! –Dan Dan: Can you please give me your "Magic Links" for both the Windows and Mac versions of the 2016 SEA VM? I have them uploaded to my Amazon Cloud Drive. But they seem to be pretty unstable in download completion. Thanks. Greg

Link to this post | posted 13 Jan, 2016 19:35

GregFrederick@letu.edu

Dan Russell
Sharon Isern
Can students get access to the VM 2016 via seaphages.org? Or do they have to be logged in as faculty? We're in class right now. Thanks!!

Because of the software included, we couldn't post it totally publicly. And since there's no "registered student" accounts on PhagesDB, there's no way to give them access to the protected page.

That said, I'll email you (and any other faculty who want it) the magic link!

–Dan

Dan: Can you please give me your "Magic Links" for both the Windows and Mac versions of the 2016 SEA VM? I have them uploaded to my Amazon Cloud Drive. But they seem to be pretty unstable in download completion.

Thanks. Greg smile

Posted in: SEA-PHAGES Virtual Machine → Student download of 2016 VM

Link to this post \| posted 13 Jan, 2016 19:34
GregFrederick@letu.edu	Dan Russell Sharon Isern Can students get access to the VM 2016 via seaphages.org? Or do they have to be logged in as faculty? We're in class right now. Thanks!! Because of the software included, we couldn't post it totally publicly. And since there's no "registered student" accounts on PhagesDB, there's no way to give them access to the protected page. That said, I'll email you (and any other faculty who want it) the magic link! –Dan Dan: Can you please give me your "Magic Links" for both the Windows and Mac versions of the 2016 SEA VM? I have them uploaded to my Amazon Cloud Drive. But they seem to be pretty unstable in download completion. Thanks. Greg

Link to this post | posted 13 Jan, 2016 19:34

GregFrederick@letu.edu

Dan Russell
Sharon Isern
Can students get access to the VM 2016 via seaphages.org? Or do they have to be logged in as faculty? We're in class right now. Thanks!!

Because of the software included, we couldn't post it totally publicly. And since there's no "registered student" accounts on PhagesDB, there's no way to give them access to the protected page.

That said, I'll email you (and any other faculty who want it) the magic link!

–Dan

Posted in: SEA-PHAGES Virtual Machine → Student download of 2016 VM

Link to this post \| posted 15 Sep, 2015 22:14
GregFrederick@letu.edu	Hmmmmm…. The plates with dilutions of putative phage stocks looked pretty much all the same and exactly like the plates with cells alone and with PB+cells. But I guess it is worth a try to do a quick spot test of each enrichment culture before we throw the entire lot out. We sent students back out with new collection kits. It seems that most did not actually follow (or possibly hear) our instructions on what the conditions of the most probably site would look like. Many collected soil from areas with little vegetation cover and areas lacking decaying matter such as wood mulch that sounded like they would not remain moist for long in the Texas heat and sunshine. But I think we should try a spot test before we discard the original enrichment stock isolates just in case. Thanks for the idea. Edited 15 Sep, 2015 22:15

Link to this post | posted 15 Sep, 2015 22:14

GregFrederick@letu.edu

Hmmmmm…. The plates with dilutions of putative phage stocks looked pretty much all the same and exactly like the plates with cells alone and with PB+cells. But I guess it is worth a try to do a quick spot test of each enrichment culture before we throw the entire lot out.

We sent students back out with new collection kits. It seems that most did not actually follow (or possibly hear) our instructions on what the conditions of the most probably site would look like. Many collected soil from areas with little vegetation cover and areas lacking decaying matter such as wood mulch that sounded like they would not remain moist for long in the Texas heat and sunshine.

But I think we should try a spot test before we discard the original enrichment stock isolates just in case. Thanks for the idea.

Edited 15 Sep, 2015 22:15

Posted in: Phage Discovery/Isolation → Smeg grown in 7H9 Complete W/ or W/O Tween for enrichment???

Link to this post \| posted 15 Sep, 2015 16:15
GregFrederick@letu.edu	Dan Russell Yep, that looks like D29! Glad it's working. Me too. Still a little trouble shooting to be done as we did not see any likely plaques on the student isolate plates (11 post-enrichment plate sets analysed this morning. About 18 to be looked at more closely this afternoon.) Out of those 11 sets, only 1 had anything even remotely resembling plaques and those were 2 putatives from 50uL of an undiluted enrichment sample. If those are real, we calculated the PFUs of that stock to be ~40pfu/mL. Which seems very unlikely! But they were small plaques which might be predicted to have a smaller burst size. IDK. Edited 15 Sep, 2015 16:29

Link to this post | posted 15 Sep, 2015 16:15

GregFrederick@letu.edu

Dan Russell
Yep, that looks like D29! Glad it's working.

Me too. Still a little trouble shooting to be done as we did not see any likely plaques on the student isolate plates (11 post-enrichment plate sets analysed this morning. About 18 to be looked at more closely this afternoon.) Out of those 11 sets, only 1 had anything even remotely resembling plaques and those were 2 putatives from 50uL of an undiluted enrichment sample. If those are real, we calculated the PFUs of that stock to be ~40pfu/mL. Which seems very unlikely! But they were small plaques which might be predicted to have a smaller burst size. IDK.

Edited 15 Sep, 2015 16:29

Posted in: Phage Discovery/Isolation → Smeg grown in 7H9 Complete W/ or W/O Tween for enrichment???

Link to this post \| posted 15 Sep, 2015 13:52
GregFrederick@letu.edu	Well…. Well… Welll… I should have gone back into the lab prior to posting here and talking with Kevin on the phone this morning. On Friday afternoon, after 24 hours growth, I did not see any plaques in the positive control plates. Fred looked yesterday and said he hadn't seen anything. But this is what we see this morning! Sorry for the stress. Thanks for the trouble shooting. You guys are great! All systems go! Edited 15 Sep, 2015 16:09 1.17Mb

Link to this post | posted 15 Sep, 2015 13:52

GregFrederick@letu.edu

Well…. Well… Welll…

I should have gone back into the lab prior to posting here and talking with Kevin on the phone this morning.

On Friday afternoon, after 24 hours growth, I did not see any plaques in the positive control plates.

Fred looked yesterday and said he hadn't seen anything. But this is what we see this morning!

Sorry for the stress. Thanks for the trouble shooting. You guys are great!

All systems go! smile

Edited 15 Sep, 2015 16:09

Posted in: Phage Discovery/Isolation → Smeg grown in 7H9 Complete W/ or W/O Tween for enrichment???

Link to this post \| posted 15 Sep, 2015 12:32
GregFrederick@letu.edu	We have been growing the liquid cultures in 250ml and 1L baffled Erlenmeyer shaker flasks. Those have either Al foil or steel slider caps. We have the disposable vented flasks in stock now. So for time sake I was going to use those today. Suggestions? (P.S. Thanks for the quick replies!) Edited 15 Sep, 2015 12:32

Posted in: Phage Discovery/Isolation → Smeg grown in 7H9 Complete W/ or W/O Tween for enrichment???

Link to this post \| posted 15 Sep, 2015 12:13
GregFrederick@letu.edu	Thanks Debbie. I am in the process of remaking all the buffers and solutions. I allowed an undergrad to make them and so I am not certain I can trust the CaCl2 and PB solutions. I have played with a lot of E. coli phages and some from other Gm+'s in the past. The shaker cultures were started from single colonies. Since we did not have the 7H9 powder when we first cultured those plates I substituted Brain Heart Infusion agar. After 24hrs, there was a haze on the plates but no discernible colonies. After 2-3 days there were visible rough textured colonies, all of one apparent type. The other caveat is that Fisher was back ordered on Carbenicillin so we were trying to culture without it. It came in on Friday. So we will have it in the culture media from now on. There was some obvious contamination on the student plaque plates. So this should at least help with some of that issue. CLARIFICATION QUESTION: I want to be certain I am doing this correctly. I know the Top Agar and Luria agar should not have Tween 80. Am I correct that the overnight shaker culture used for plaque plating should NOT have tween 80 in it? The Smeg will not clump without the Tween 80 in the 7H9 broth???

Link to this post | posted 15 Sep, 2015 12:13

GregFrederick@letu.edu

Thanks Debbie. I am in the process of remaking all the buffers and solutions. I allowed an undergrad to make them and so I am not certain I can trust the CaCl2 and PB solutions.

I have played with a lot of E. coli phages and some from other Gm+'s in the past.

The shaker cultures were started from single colonies. Since we did not have the 7H9 powder when we first cultured those plates I substituted Brain Heart Infusion agar. After 24hrs, there was a haze on the plates but no discernible colonies. After 2-3 days there were visible rough textured colonies, all of one apparent type.

The other caveat is that Fisher was back ordered on Carbenicillin so we were trying to culture without it. It came in on Friday. So we will have it in the culture media from now on. There was some obvious contamination on the student plaque plates. So this should at least help with some of that issue.

CLARIFICATION QUESTION: I want to be certain I am doing this correctly. I know the Top Agar and Luria agar should not have Tween 80. Am I correct that the overnight shaker culture used for plaque plating should NOT have tween 80 in it? The Smeg will not clump without the Tween 80 in the 7H9 broth???

Posted in: Phage Discovery/Isolation → Smeg grown in 7H9 Complete W/ or W/O Tween for enrichment???

Link to this post \| posted 14 Sep, 2015 17:55
GregFrederick@letu.edu	Just FYI. We got zero visible plaques using a Smeg culture grown with Tween 80 in the shaker culture. only the liquid culture had Tween 80. Plates and top agar did not. I am not certain if this was the problem or if there may have been problems with the phage buffer or CaCl2 solution concentrations. We did not even see plaques on the D129 plates. I am remaking everything today to try and troubleshoot before we have the entire class proceed and burn up a lot more plates and reagents. Thoughts? QUESTIONS: Am I right that even the overnight Smeg culture should be grown WITHOUT Tween 80 for phage plaquing?

Link to this post | posted 14 Sep, 2015 17:55

GregFrederick@letu.edu

Just FYI. We got zero visible plaques using a Smeg culture grown with Tween 80 in the shaker culture. only the liquid culture had Tween 80. Plates and top agar did not. I am not certain if this was the problem or if there may have been problems with the phage buffer or CaCl2 solution concentrations. We did not even see plaques on the D129 plates.

I am remaking everything today to try and troubleshoot before we have the entire class proceed and burn up a lot more plates and reagents.

Thoughts?

QUESTIONS: Am I right that even the overnight Smeg culture should be grown WITHOUT Tween 80 for phage plaquing?

Posted in: Phage Discovery/Isolation → Smeg grown in 7H9 Complete W/ or W/O Tween for enrichment???

Link to this post \| posted 09 Sep, 2015 16:17
GregFrederick@letu.edu	Thanks Kevin. I appreciate the email too! I am so glad you all are there! gf

Posted in: Phage Discovery/Isolation → Plaque Bottom Agar - Recipe???

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