Link to this post | posted 18 Sep, 2018 20:46
debbie	Maja is an Arthrobacter phage. Gene 27 has transmembranes identified. I do not know what the function is other than "membrane protein". Maja has 2 integrases - a serine and a tyrosine integrase. It also looks like it has most of the pieces of the lambda repressor system, though most are labeled as helix-turn-helix DNA binding proteins for now.

Link to this post | posted 17 Sep, 2018 15:55
debbie	Jason, The simplest explanation is one of the following: a. you have a non-pigmented mutant b. it is a contaminant (and no longer G. rubi) I would recommend going back to the frozen stock. debbie Edited 17 Sep, 2018 16:10

Link to this post | posted 14 Sep, 2018 14:14
debbie	I use the filtered sample. Evaluate this choice if the number of plaques is <10 in the spot. In this case, I MAY carefully consider picking from the spot. I would also change the volume that I plate from the enrichment. The second consideration is if the spot is contaminated. You may want to refilter or you may have to go back to the original sample and carefully filter. Good luck. debbie

Link to this post | posted 13 Sep, 2018 19:04

debbie

Nikki, Hi. I have no really data to share, but here is my experience. 1. How wet are the plates? sometimes if there is a lot of cold condensation on the surface of the plate, when you add top agar it stays a soup (or a slippery mess). You can avoid this by keeping your plates at room temperature (we do). There are no antibiotics in our PYCa plates, so no harm… 2. The hotter the top agar, the better. (I have no explanation for that, but I have less agar problems when my top agar is on the hot side of things.) 3. Yes, I think the room temperature, but more likely the humidity, plays a factor. I saw this most often when I was in classrooms and in a hurry to get the plates off the benches. I have not data to share to support that. Do you have students vent (i.e. crack them a bit) their plates when drying? I always vent my plates for a short time. It is unlikely that you have bunsen burners in the area, but having a bunsen burner burning in the background sometimes helps (may be totally unsafe in a student lab environment, so it is more important to stay safe). again, this works because the air is dryer.) All I've got for now. Good luck! debbie

Link to this post | posted 10 Sep, 2018 19:07

debbie

Denise, I knew I knew something about this but it took me a few prompts (thanks, Chris) to recall anything. It might be helpful to go to DMDB folder of the DNA Master application folder. there should be a GBS folder for each project. if you have no projects, you should not have any folders, so I am not sure that will fix anything. In July, I had the same problem. Here is what Jeffrey had me do to fix it. However, I couldn't fix and and sent him files. He eventually got it fixed, so I am not sure this will help. According to Jeffrey, the BLOB error means: That means the database tables are not synchronized; each table has multiple files, and premature closing of a program or other database failure can cause them to get out of sync. Close DNA Master Go to the program directory and open the Helper directory Run the dtutil32 program. This is an immensely useful program. Repeat steps below for any database errors. Click the “By Directory” button In the dialog, go to the DMDB directory and open the GenbankProjects.db file Click the Verify button Click the Rebuild button Note any reports that the verification or rebuild was not successful. If so, repeat steps 7 then 6 on the file. If there is corruption, they should rebuild without error, so no errors reported does not mean nothing is working. Repeat steps 4-8 for the GenBankAuthors and GenBankReferences files Restart DNA Master Those are the DB files for GenBank projects and are the most likely files with the out-of-sync entries. Me gain - So I tried this, like I said and couldn't make it work. since then, though, I have corrupted a file or 2. I sought out the GBsubmit folder and deleted the specific folder for a genome. When I did this, I corrupted an 'old' project, so i think it is still applicable to your problem… I know this isn't very helpful, but maybe in wiser hands than mine it will work… You are welcome to call. debbie Edited 10 Sep, 2018 19:10

Link to this post | posted 05 Sep, 2018 13:12

debbie

Jeremy, I think the simplest way to think about that is that "old" bacteria would be a selection bias. Because of the large number of phages that plaque on "old" bacteria, I don't think that is what would stop you from finding phages. But, it will stop you from finding phages we haven't ever found before (because we may have been using "old" bacteria). We have an anecdotal report from Northwestern College that they found a Mycobacterium phage singleton because they selected for tiny plaques and they were using "fresh" smeg. It is an N of 1, but still interesting. Good microbiology says to not use old cultures. So it will never hurt to grow them weekly. The most likely causes of not finding phages from enriched samples are: The smeg is clumpy. Use a Tween starter culture, so you start with individual cells. Keep shaking the culture for 3 days (you can use it as soon as you have a dense enough culture, but then put it back on the shaker. Do not let it sit still on your bench until you are sure cells have reached stationary phase. Otherwise a biofilm will form and clump your culture. Enrichment time is not long enough (or too long). How many days are you enriching? The number of phages found increases with the right timing. Are you enriching at 37? Plate at the same temperature as your enrichments. When (and how often) do you check your plates for plaques? Sometimes, the plaques are so turbid, they can overgrow to look like an uninfected bacterial lawn. Other times, a plaque - especially from the original sample - takes a few days longer to 'appear'. Good luck, debbie

Link to this post | posted 28 Aug, 2018 20:56
debbie	Kayla, I think everything looks as I would expect. Do you think it is all correct now? debbie

Link to this post | posted 27 Aug, 2018 18:56
debbie	Kayla, Would you do dilutions of D29 and not just a single spot? You streak from P1FF streak from stock #2 looks like smeg. When you see 4 day colonies, are they dry and wrinkled? (as in, not smooth and glistening?)

Link to this post | posted 23 Aug, 2018 19:59
debbie	Be careful! The auto-annotation predicts forward strand proteins in the middle of the genome. The proteins in the reverse strand are better supported.

Link to this post | posted 23 Aug, 2018 14:38
debbie	At this time (8-23-18 ), we felt it prudent to call the DNA binding proteins around the integrases "helix-turn-helix DNA binding proteins", even thought 2 of them are hitting Lambda's CI. (Genome used in this evaluation are Nandita and Ryan). Edited 23 Aug, 2018 14:39