Link to this post | posted 06 Jul, 2023 19:08

mdgainey

Can anyone advise on whether the dry uranyl acetate waste generated from the EM staining process is considered exempt waste because of the very small dry amounts of waste, or if it has to be disposed of via a radioactive waste route? I am getting ready to write an SOP for staining using uranyl acetate at my institution and want to make sure that I handle the waste properly/prepare for the costs ect.

Link to this post | posted 21 Jun, 2023 18:06
mdgainey	Here are some screen shots of the demo in power point. I could not attach the demo files. 1.53Mb

Link to this post | posted 21 Jun, 2023 17:54

mdgainey

We did end up sending identically stained grids (Cluster M3 bacteriophage Nanosmite) to 3 companies that sell SEM's with STEM detectors Thermo-Fisher, JEOL and Zeiss and they each generated images of them as a part of their demo for us. We got the best results with Zeiss, but also some nice results with the Thermo-Fisher set up. I will attach these images (just keep flipping through until you see the phage samples. The SEA team can chime in here bc I am not an EM expert, but I thought at first blush the images that they obtained look good enough to meet the programatic standards, but I don't think that they will be as good as some of the TEM images that I have seen… We ended up going with the Thermo-Fisher model with a STEM3+ detector cat # 1091077 (detector is ~$27k) just simply due to cost differences. It took a long time to get them to come out and set it up for us (not happy with their service=( but I just recently had the chance to try it out with the same samples (now 2.5 years old). I have attached my first image, which is not as good as their demo images possibly due to sample age, our set up, or the fact that I don't really have any microscopy skills. As I get more comfortable I will post some additional images that I obtain so folks can get a sense of what this STEM setup can do. 3.22Mb

Link to this post | posted 20 Jun, 2023 19:25

mdgainey

This is a fun paper that I did in phage bioinformatics this year that my students enjoyed. We use code 11 (bacterial, archael and plant plastid code) for our annotations because TTG (UUG) and GTG (GUG)are used as start codons. This paper demonstrates the some human microbiome phages use code 15. In code 15 TAG(UAG) is not used as a stop codon and instead will code for the amino acid Glutamine. I thought it was very relevant to what we were doing and shows how much "better" these genomes look after annotation when the proper code was used. https://www.nature.com/articles/s41467-022-32979-6

Link to this post | posted 12 Nov, 2022 19:21

mdgainey

I have had good luck with some but not all of my student's G. rubri phages by taking 4mL of the students HTL pretreating with DNase/RNase, then spinning for 10,000rpm for 40 min at 4 degrees C as described in the instructors guide, and pulling off enough of the supernatant to end up with ~500ul of the end. I then immediately add EDTA ect. It says in the guide that this method is not as consistent as the ZnCl2 protocol, which may be true but it is easy and normally bumps up a few of my students DNA concentrations nicely without me having to have the students make a bunch of new stocks. I would be happy to send you the full protocol I use if you like. You could also do the spin, and then try phenol/chloroform extraction, instead of the wizard kit if you are comfortable with that.

Link to this post | posted 20 May, 2022 14:16
mdgainey	Thanks Debbie, I will leave them in my GenBank submission for now. M

Link to this post | posted 10 May, 2022 13:59
mdgainey	Hi Rick, I am going to look into some of the these repeats with my students this summer. They also have a different repeat towards that middle of the genome where there are a lot of membrane proteins, and there are also a lot of gaps. You can also find them using MEME, and this generates a nice output to look at. I am going to give this another eyeball before I submit my final DJ annotation.

Link to this post | posted 28 Apr, 2022 17:21

mdgainey

I am working with a cluster DJ phage named Vardy this semester, that seems to have a surprising amount of membrane proteins (they called 16 initially), with many clustered together in the same area of the genome. There are also some with signal peptides in this area (predicted by signal P and topcons). I looked up the past signal peptide post and it seems that these we were not calling, but given that the idea here is just to alert someone about interesting areas of the genome where these membrane and potentially membrane associated proteins are, can I call these membrane proteins as well just to denote their presence for now, or can we add a signal peptide containing protein to the choices on the list? I will have some students look into this area a bit more this summer as it is also chocked full of interesting repeat sequences.

Link to this post | posted 09 Sep, 2021 17:34
mdgainey	I received a new computer this summer, and am currently trying to install the 2017VM. I thought I was good to go, but when I launch the VM I get an error: attempt to read or write outside of disk 'hdo' Does anyone know how to fix this?

Link to this post | posted 23 Jan, 2021 16:19
mdgainey	Thanks Debbie and Kirk,I missed this on the list due to not doing control F with the capital H in holliday. I felt like it should be there, and it is is….. Edited 23 Jan, 2021 16:19