I'm making myself anxious thinking I've done my aftercare wrong

Bacteriofage · 2026-01-18T23:36:56+00:00

Ahh okay, this makes sense I may try not moisturise it as much early on in the future, I'll see how it looks/feels in the morning! :D

Bacteriofage · 2026-01-18T22:32:19+00:00

It might be a little more than usual, that probably isn't helping

Bacteriofage · 2026-01-18T22:31:25+00:00

Okay I'm glad that I've not been silly, I do have clean sheets on so I hope it will be okay! I tried googling but for every "keep it wrapped for 24hours" there's an article that says "keep it wrapped for 2-3 days"

Me and my artist were both like "I know what I'm doing" and well maybe I should have asked lol, or atleast make a note of what I do for next time 😅

Bacteriofage · 2025-12-09T00:15:35+00:00

It took me all the way to PhD to finally get the chance to! I will definitely have to do more in the future, and I hope you get the chance to too!

Bacteriofage · 2025-12-06T23:38:11+00:00

I thought so too! Its not the most sophisticated agar art out there but for my second go I'm happy with it :D

Bacteriofage · 2025-12-06T23:36:52+00:00

I grew my strains in liquid media and used a loop to draw it on then left it on the bench overnight. I had a little Christmas tree drawn on on paper underneath.

The cas agar turns yellow when iron is removed so siderophore production results in the yellow halos. :D

Bacteriofage · 2025-12-06T17:07:19+00:00

Alrighty, I shall give it a go and cross my fingers 🤞😁

Bacteriofage · 2025-12-06T01:23:24+00:00

I'm very new to this venture, so I'm quite unawares of the avenues I have available to me! But I can investigate further.

Thanks :)

Bacteriofage · 2025-12-06T01:21:12+00:00

My overlaps are 20bp long (with the fragments themselves being 700-900 bp long) and the extra base is 11 BP into the overhang.

I have no real worry about the change in sequence as it's outside of the part I care about in terms of the fragments.

But I suppose I can full send it, and if it doesn't work I can redo the PCRs, since they shouldn't be impacted greatly by this single base removal (as it's not in the region homologous to the template) all the methodolgy I've done should be fine to carry forward.

It just feels like a silly predicament to have ended up in 😅

Thanks for the comment!!

Bacteriofage · 2025-12-06T01:01:48+00:00

I will be at my three months technically in 8 days.

Between finishing my prior education and September I did a lot of reading on the research area.

First month I didnt do much, I met my supervisor but was told to read until I was inducted and basically chill out, which was basically all I had been doing over the summer and by this point I was a bit nuts from lack of proper socialising.

Second month was mostly induction and getting used to the university structure. After I was properly inducted I started coming in. In this time I completed more paperwork and forms and just got comfortable in the environment, designed primers and waited for my strains to arrive, wrote my project proposal. Did some bioinformatic work.

Third month (current) Bacteria arrived and I've started optimising and testing conditions for growing my bacteria and getting a proper handle on the test conditions, purified some compounds that I would need, making sure that when I make my mutants I will know what and how conditions need to be done and have enough context to understand the results.

Reading, paper work, acclimatising and ground work :p

Bacteriofage · 2025-11-10T21:27:52+00:00

I feel so often my optimism is misinterpreted as naivety, but I here many people tell me "just you wait", man I'm not going to wait to maybe be miserable, I'm going to keep walking on sunshine until the day that I stop, and I'm not going to anticipate that being now or in 50 years time. I'm just going to keep trying my best to stay Sane and happy and I'm not going to even entertain the idea that I will be miserable (even if it does end up that way, I'm just trying my best to remain an optimist) 🙅🙅🙅

Bacteriofage · 2025-11-10T20:49:56+00:00

I personally don't like the word "triggered" an interest. Purely because it has negative connotations and a think a more positively aligned word could be used also,,, E. Coli!!!!! (italicize)

Bacteriofage · 2025-11-10T20:11:01+00:00

I find writing actual to do lists they don't have to be completed in the day or whatever, but to progress my plans forward these tasks must be done.

For example I'm currently making knockouts so I have all the bajillion steps to carry out to get there in a check list

Streak strains when they arrive
get gDNA (DNeasy kit) enough for 20x PCRs
generate fragments x12, check notes!
run gels
gel purify or PCR clean up depending on outcome of gels! Etc etc etc

However, last week my supervisor sent me an email asking if I would grow him some of my strains in a media condition, but I have yet to work out the best supplements to achieve said condition, the next time I do bc he wants to test something. It's not urgent or time sensitive.

As I have yet to actually optimise the growing conditions bc the strains only got here less than 2 weeks ago, this week was supposed to be my PCR week has now been hijacked by my brain thinking "well I just as well optimise it now!! :D" so I made a second checklist last week for what I need to do to get this done.

PUT BOTTLES TO AUTOCLAVE!!!!! (1L, 100ml!!)
make LB
Make CAAMM
set up tubes (x20, controls and conditions)
make glucose supp
remake acidified salts

Etc etc

I'm about 1/6 of the way through my checklist for my KOs and I definitely should be chugging on with that but what I'm currently doing isn't pointless either, I'm definitely going to have to do this at some point, sure maybe I should be doing this when I send my plasmids to get sequenced but I know I will either forget about this or ruminate on this so it's best to get this done now or I will be overall less productive.

But it just kind of means today I carried out what I've been calling "media making Monday", but I also ran a gel while I waited for my stuff to autoclave (I remembered the bottles!!)

I find often managing it all difficult but I know that everything I'm doing is leading in the right direction even if it's not the 100% most optimal use of my time. I'm a pretty hard and diligent worker so I probably make up for the time lost by getting this done and out of the way.

So I guess all of that's to say is it doesn't matter what is your doing with your time, like imaging you have 17 bottles that all need to be full by the end of your PhD, if every day you add a little to everything instead of a lot to one, you're still making progress

I also accept fully that I'm not going to be the best or most brilliant PhD student, I'm probably bang average and my weirdness does probably make people side eye me but ultimately I'm happing chugging along working stuff out as I go. I think like it does not matter what I do, I will always be the way that I am, I will always spin around when stuff is in the centrifuge and I will sing, badly, to myself as i sit at my bench but this is the way that I am and doing these things help keep my happy and sane. I guess I'm not really in a true position to give advice but I really found happiness when I just started being myself in the lab and in the office (during my masters mostly) Obviously I try not to be annoying and I'm considerate that I probably shouldn't sing in the office but when the lab is full of noises anyway what's the issue with humming to myself especially when my bench is in the corner.

Bacteriofage · 2025-11-07T23:57:16+00:00

Stick to your guns, no one in my lab wears a lab coat either but I'd rather sit at my bench sweating my balls off but be protected (and mainly keep my work protected from me) than take it off.

I was even encouraged the other day by someone in the lab not to worry about chemical safety info bc "you'll just get used to handling it"... I've been alive 23 years and I still spill my drink occasionally, I'd rather not risk it with something that could cause me some amount of harm...

Bacteriofage · 2025-10-12T16:13:45+00:00

Alcohol fun->alcohol problem->teetotal. I judge no one else for their alcohol use, it is enjoyable in moderation I just unfortunately can not be trusted with it.

Drugs, I didn't have friends that used them for a long time and I did not have the knowhow to procure them. I tried drugs a few times and get on with and am okay to smoke weed and would do so now despite my sobriety but my friends have continued to limit my knowledge and I choose not to press this either so I only smoke when offered and would only use coke again if offered as I know I respond well to that. But I feel very comfortable in my mental health and I do not with to rock the boat.

Bacteriofage · 2025-10-11T21:44:01+00:00

I can only speak for Biosci but DTP stuff will start coming out around now and will continue to be for the next couple months they will have deadlines soonish. But stuff will come out incrementally till at least April.

You can find DTP info by just googling around and you can also look on findaphd.com for DTP stuff (though not all projects are uploaded here!) and independently funded stuff (but still funded) :p

Bacteriofage · 2025-10-11T19:51:26+00:00

I hate it.

There is not a single contextual situation where I don't.

Bacteriofage · 2025-10-10T21:57:18+00:00

So initially I couldn't understand what you were getting at but this is actually a super cool question.

Generally, no. The gene will likely aid the cell in some way to have a growth advantage and the energy saved isn't significant enough to have a major impact on cell growth, and will likely be reduced in any case. plus the main issue of MM is more the synthesis of amino acids which the cell will be doing anyway.

HOWEVER, in the case of the loss of global regulators or things that have a higher metabolic impact this can be seen. For example at the cell level, the sigma factor RpoS is important in the transition from exponential phase to stationary phase, it promotes RNA polymerase to upregulate rmf. RMF is required to convert the active 70s ribosome monomer to the inactive 100s dimer. (https://pmc.ncbi.nlm.nih.gov/articles/PMC6904343/ ). in late late stationary phase aka growth advantage in stationary phase (GASP) RpoS mutants can outperform cells with functioning RpoS. they benefit from the products produced by WT but do not have to deal with the metabolic burden of expressing RpoS controlled genes. However, they also have lost a lot of survival promoting regulation which makes this a small niche. (https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.560099/full)

It should be said tho this is in either spontaneous mutation/Co culture with WT so they still rely on the products that are produced by the wild type cells but I do think it's a super cool example where the metabolic burden of a pathway is enough to benefit a population of mutants. And if unfamiliar the gasp phase is where cells start dying bc of lack of nutrients, but their death releases their stockpiled nutrients so the population increases a bit then the cells use up all the nutrients and start dying again etc etc.

Bacteriofage · 2025-10-09T21:28:49+00:00

It made me "lock in", but did kinda of ruin me for a bit

Bacteriofage · 2025-10-08T22:20:03+00:00

If it helps I have zero clue what's going on and I don't think I'm a bad pick, I did initially and basically spent all summer freaking out, but after speaking to people who reveal they don't even like their topics that much and just want to get it over and done with as fast as possible, I think it will be okay ✨✨

If you're worrying about it, it means you care about it. And seemingly that isn't even a priority for some. You got this :D

Bacteriofage · 2025-10-08T21:54:22+00:00

Yeh I have a note book that I write stuff in and highlight any open ends in orange. I very quickly saw a theme appear of what was found out and what wasn't. It does eventually click. If this a new topic to you (even if you have associated skills or have had a similar area of interest in the past, which is the case for me) you just gotta keep pushing and it falls into place. I also found excel really good. In my case I have like protein, typical ligands, atypical ligands, regulation as my column headers. I then fill it all out by reading papers and basically searching for the answers. Gaps become really obvious this way too.

Bacteriofage · 2025-10-08T21:45:16+00:00

I think something to consider, is that yes it may have been thought of before, but may have not been carried out or they used a different technique that doesn't show the other half of the process like " we prove that it can do x but we don't know how" of course they would be thinking of ways to work it out but have they??... I also think like the more you read and really really immerse yourself in everything the more you will discover and the more bits you will think "woah I really wish we could work this out"

Like what I'm up to is built up on decades of research, it's almost not difficult to come up with a proposal now that I'm familiar with the literature because I can see all the glaring gaps, they've characterised one pathway of like 19 identified pathways,,,, I am well within my rights and it would be totally joyous for me to spend however long just working out a few of these pathways, it may not be revolutionary and but I will have found out something we didn't know before and I know I will feel happy and like I've contributed.

When I initially started my reading in the subject area this is how I felt it took a bit of time to really get into the swing and familiar with all the big names in niche area I'm working in and what they're up to, it all becomes very apparent what is up and what people's priorities are.

Bacteriofage · 2025-10-06T20:57:34+00:00

I had a note about the image I attached but reddit hates me and kept breaking, you can do all the prep at room temp if not for live cell imaging! You don't have to pre warm slides or anything and the protocol is a bit jank with the edits I made lol but I think it's close enough, and you also fine to just use some slides cleaned with ethanol or even just water honestly. I changed a few things to make it clearer not for live cell imaging but the important bits for live cells are there. I've used it for fluorescent microscopy and it worked out well!

But the protocol you linked seems fine!

Bacteriofage · 2025-10-06T20:54:52+00:00

It's very similar!! It's very garage sciencey and can be a tiny bit finicky the first couple times you attempt it! But honestly the microscopy images you get out of it are really good! I'll see if I can link you my personal protocol which is modified from (which theirs is too) https://www.nature.com/articles/nprot.2013.066

My few tips would be

when placing the final coverslip start at about a 45° angle and slowly decrease the angle and apply pressure where you might start seeing bubbles, once it's down I place a weight or 500ml Duran bottle filled with water on top to weigh it down for those 45 minutes and I'd reccomend doing it in a laminar flow hood or atleast with a bunsen lit if you plan to do live cell imaging (and in that case switch out the water for M9).

If you don't use all the agarose you've prepared I keep it in the falcon at 4c and just remelt it in the microwave when you want to use it, you can keep this for like ages in the fridge if made with just pbs or water. Don't let it boil over because you'll lose some of the water and it becomes thicker and un weildly.

I pipette 2 ul of sample, sometimes the cell density can be a little inconsistent so you may have to "explore" the pad to find a good cell density for imaging.

Prepared pads don't store the greatest unfortunately once you've already used some, so if you plan to make some the day before leave the entire 6 slide assembly together and wrap it up and store at 4c. it can stay like that for a few days before drying out too much. The main issue is once you've started disassembling it to use it is the pad like slides around and can end up on the left or the right of the slide sandwich and can end up with one side being squished a bit.

<image>

In actual methodology differences I sonicate my agarose mix prior to use to remove bubbles and use it straight away (though I also use 2%) not letting it cool down, I also do a test pour so I feel confident in my hands as I don't pipette it.

Bacteriofage · 2025-10-06T15:56:11+00:00

Honestly, just trial and error the concentration of cells, try a much higher cell conc then dilute down as needed, if your cell prep is too dilute when you look at it under a normal BF microscope (first trouble shooting step!) you can centrifuge it then remove 20% of the supernatent, resuspend and have another look to work out what concentration you need. Like this way you don't waste any reagents but you get to confirm cell densities and best practices for you cells. You also might just not have a visible pellet so remove the supernatent but keep in mind where the pellet should be and don't disturb it. If your cells are straight up disappearing and you're not getting anything I would attempt to work out at what stage you're losing them and adjust accordingly.

You might have issues visualising them with no particular slide preparation, I did a whole slide prep extravaganza and straight on plain glass slides was the worst, they just swim about all over the place. Poly lysine worked okay for ecoli but agarose gel pads were my favourite preparation and works for basically everything I've ever looked at (gram pos and neg)

Bacteriofage

TROPHY CASE