SEA-PHAGES | All posts created by mdgainey

Link to this post \| posted 14 Oct, 2017 12:04
mdgainey	Hi all, hope your semester is going well. We are currently working with phages isolated from Microbacterium foliorum. I have thus far observed lower DNA yields with these phages than with phages from Mycobacterium at comparable titers. I have been able to get decent yields by peg precip/pelleting, but have noticed for about half of my phages that I am likely precipitating a nuclease of some type (in preps done without DNase/RNase treatment that are not for sequencing). Hopefully an sds/edta/protK-55degree incubation before the Wizard kit will remedy this problem. I also think that some of these phages are less stable at 4 degrees than the phages from Mycobacterium, so perhaps preps from very fresh stocks may be best? Has anyone else observed this? I also wanted to perhaps make an effort to standardize the restriction enzymes digests that we are using and uploading to phagesdb.org. Many of the Microbacterium phages on phagesdb do not have digest pics, and the ones that do often have a single digest shown, but I am not sure what enzyme has been used. In the guide HaeIII, NspI, and XceI are suggested. How are these digest looking for those that have tried these enzymes, and what conditions are you using to run your gels. I am assuming HaeIII is done on a very high percentage agarose gel and would love any tips that anyone has about running these. Thus far my phages fall into two groups, ones that are cut by the traditional enzymes (Bam,cal,eco,hae, hind) and those that are only cut by HaeIII. Lastly, I have found the phageenzyme tool to be very useful in the past for preliminary cluster assignment of my phages and would like to continue using it for the phages isolated from Microbacterium but many of the sequenced Microbacterium phages are not yet popping up when I use this tool. Is the tool updated periodically, or should I request to have certain phages added?

Link to this post | posted 14 Oct, 2017 12:04

Hi all, hope your semester is going well. We are currently working with phages isolated from Microbacterium foliorum. I have thus far observed lower DNA yields with these phages than with phages from Mycobacterium at comparable titers. I have been able to get decent yields by peg precip/pelleting, but have noticed for about half of my phages that I am likely precipitating a nuclease of some type (in preps done without DNase/RNase treatment that are not for sequencing). Hopefully an sds/edta/protK-55degree incubation before the Wizard kit will remedy this problem.
I also think that some of these phages are less stable at 4 degrees than the phages from Mycobacterium, so perhaps preps from very fresh stocks may be best? Has anyone else observed this?
I also wanted to perhaps make an effort to standardize the restriction enzymes digests that we are using and uploading to phagesdb.org. Many of the Microbacterium phages on phagesdb do not have digest pics, and the ones that do often have a single digest shown, but I am not sure what enzyme has been used. In the guide HaeIII, NspI, and XceI are suggested. How are these digest looking for those that have tried these enzymes, and what conditions are you using to run your gels. I am assuming HaeIII is done on a very high percentage agarose gel and would love any tips that anyone has about running these. Thus far my phages fall into two groups, ones that are cut by the traditional enzymes (Bam,cal,eco,hae, hind) and those that are only cut by HaeIII.
Lastly, I have found the phageenzyme tool to be very useful in the past for preliminary cluster assignment of my phages and would like to continue using it for the phages isolated from Microbacterium but many of the sequenced Microbacterium phages are not yet popping up when I use this tool. Is the tool updated periodically, or should I request to have certain phages added?

Posted in: Microbacterium → Restriction Enzyme Digests

Link to this post \| posted 06 Feb, 2017 16:23
mdgainey	Hi Lee, on a bit of an unrelated note, would you be willing to share your Streptomyces griseus phage hunting protocols with me? I am getting ready to order this host from ATCC. If you have any tips for success that you are willing to share I would be grateful. I have a group of students doing honors projects that I was going to have do some phage hunting in Great Smoky Mountains National park, where we have had 0 success with m. smeg.

Link to this post | posted 06 Feb, 2017 16:23

mdgainey

Hi Lee, on a bit of an unrelated note, would you be willing to share your Streptomyces griseus phage hunting protocols with me? I am getting ready to order this host from ATCC. If you have any tips for success that you are willing to share I would be grateful. I have a group of students doing honors projects that I was going to have do some phage hunting in Great Smoky Mountains National park, where we have had 0 success with m. smeg.

Posted in: Phamerator → PhamDB: Make your own Phamerator databases

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