SEA-PHAGES | All posts created by joyous726

Link to this post \| posted 26 Feb, 2016 20:27
joyous726	Welkin Pope Hi Joy, It just means that you need to streak out the potential lysogens for single colonies. Here is a youtube video (tps://www.youtube.com/watch?v=Ay2hhujTuvg) You can use sterile wooden sticks, an innoculation loop, sterile toothpicks, etc. I like sticks for students, they are easier to use and tend not to accidentally gouge into the agar surface as much. The streaking serves two purposes: one, to grow up a single clonal colony to work from, and two, to remove any exogenous phage that might be left over from the initial mesa spot. Sometimes you can get a mix of resistant cells and true lysogens. Streaking to purify cells and then working from a purified clone is good microbiology practice. Thank you! Sharon was kind enough to email me the attached protocol that basically fills in with what you have replied. What was unclear from the original protocol, initially, was a silly question like whether we needed to use 7H10 plates. We felt fairly confident, but not confident enough not to check with those with more expertise. 138Kb

Link to this post | posted 26 Feb, 2016 20:27

Welkin Pope
Hi Joy,
It just means that you need to streak out the potential lysogens for single colonies. Here is a youtube video (tps://www.youtube.com/watch?v=Ay2hhujTuvg)
You can use sterile wooden sticks, an innoculation loop, sterile toothpicks, etc. I like sticks for students, they are easier to use and tend not to accidentally gouge into the agar surface as much.

The streaking serves two purposes: one, to grow up a single clonal colony to work from, and two, to remove any exogenous phage that might be left over from the initial mesa spot. Sometimes you can get a mix of resistant cells and true lysogens. Streaking to purify cells and then working from a purified clone is good microbiology practice.

Thank you! Sharon was kind enough to email me the attached protocol that basically fills in with what you have replied. What was unclear from the original protocol, initially, was a silly question like whether we needed to use 7H10 plates. We felt fairly confident, but not confident enough not to check with those with more expertise.

Posted in: Lysogeny/Immunity → Lysogeny experiment help

Link to this post \| posted 23 Feb, 2016 16:14
joyous726	We have several students isolating lysogens and following a protocol that is on phagesdb.org (http://phagesdb.org/media/workflow/protocols/pdfs/LysogenyProtocol_3.19.13.pdf). I was wondering if there was anything additional in the way of protocols or background info that might be helpful for us. In particular, we find steps 11 - 14 a little vague and imply some working microbiological knowledge that we're a little shaky on. While we like to encourage student independence in reading protocols, we would love to have some more resources to direct them to for help. I know that other faculty have had students create stable lysogens to do immunity experiments. Is there anything that any of you have that might be helpful for us in the way of resources, additional protocols, or advice? Any help is appreciated!

Link to this post | posted 23 Feb, 2016 16:14

joyous726

We have several students isolating lysogens and following a protocol that is on phagesdb.org (http://phagesdb.org/media/workflow/protocols/pdfs/LysogenyProtocol_3.19.13.pdf). I was wondering if there was anything additional in the way of protocols or background info that might be helpful for us. In particular, we find steps 11 - 14 a little vague and imply some working microbiological knowledge that we're a little shaky on. While we like to encourage student independence in reading protocols, we would love to have some more resources to direct them to for help. I know that other faculty have had students create stable lysogens to do immunity experiments. Is there anything that any of you have that might be helpful for us in the way of resources, additional protocols, or advice?

Any help is appreciated!

Posted in: Lysogeny/Immunity → Lysogeny experiment help

Link to this post \| posted 16 Feb, 2016 17:42
joyous726	Kristen Butela Is anyone still using the cluster specific primers to assign mycobacteriophages to various clusters? I have some students who may be interested in screening some unsequenced phages we have in our archive, but I haven't been able to find the list. I'd like to spend some time on PCR and gel electrophoresis this semester, and I thought this may be an option. Is this still a useful protocol, or have we moved on to something else? We are also interested in this… we want to screen a mixed sample (F and B cluster phages), but would be interested in opening it up for non-sequenced phages, too!

Link to this post | posted 16 Feb, 2016 17:42

joyous726

Kristen Butela
Is anyone still using the cluster specific primers to assign mycobacteriophages to various clusters? I have some students who may be interested in screening some unsequenced phages we have in our archive, but I haven't been able to find the list. I'd like to spend some time on PCR and gel electrophoresis this semester, and I thought this may be an option. Is this still a useful protocol, or have we moved on to something else?

We are also interested in this… we want to screen a mixed sample (F and B cluster phages), but would be interested in opening it up for non-sequenced phages, too!

Posted in: Phage Discovery/Isolation → Cluster Specific Primers

Link to this post \| posted 29 Jan, 2016 17:37
joyous726	We have something strange going on when we compare our auto-annotated Blast file in DNA master to what's in phamerator and starterator for ShiaLaBeouf_Draft. The gene product numbers/features don't align like they do for other phage genomes. Is this because of a difference in when we did our Blasting? The files aren't corrupt (the genome size is consistent with what we started with and what is reported on phagesdb). But, gp 42 is not the same… This might be a naive newbie confusion, but… help?

Link to this post | posted 29 Jan, 2016 17:37

joyous726

We have something strange going on when we compare our auto-annotated Blast file in DNA master to what's in phamerator and starterator for ShiaLaBeouf_Draft. The gene product numbers/features don't align like they do for other phage genomes. Is this because of a difference in when we did our Blasting? The files aren't corrupt (the genome size is consistent with what we started with and what is reported on phagesdb). But, gp 42 is not the same… This might be a naive newbie confusion, but… help?

Posted in: Phamerator → Tutorial on Phamerator and Starterator Use?

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