Annual PM today. I’m the only one in our lab who runs it so it feels nice to know my labor is appreciated by someone other than me 😂😭

Sent my MSc research proposal to my PI yesterday.

This morning he sent me SMS says “Hi… please call me”.

Not knowing what have I done wrong, I panicked and thought maybe he thinks i used too much AI or something. Decided not to call him and to meet him face to face in the lab, maybe seeing my face would make him less aggressive..

He wasn’t there.

I called him, and he said “Hi, I can’t manage to open the word document, could you send me a PDF?”.

God, felt so stupid for panicking.

He never yelled at me or anything.

When is it time to throw in the towel and declare a paper unpublishable? Asking for a friend…

Bit of a rant, will keep it not very specific so as to avoid doxxing myself. I’m a PhD student in STEM, and get along very well with everyone in my group except for this one person who joined a few months ago. We don’t work together exactly, but we do work on very similar projects so we share reagents, space, equipment etc.

To be quite blunt, this person makes my life harder in a lot of ways. I find them sloppy, not cleaning up messes, forgetting to do shared lab tasks, reorder reagents etc. I do mention these things to them whenever it happens, but it seems to keep happening regardless. I do get the sense they don’t really take me seriously because I’m female and significantly younger but that’s speculation on my part.

They’re not a bad person or anything, although they have made a few comments I didn’t really like. But our work styles really don’t gel and I don’t like being around them. Obviously I try to stay out of their way as much as I can, but there’s only so much I can do that. They’ve also asked me before if they offended me in some way and I said no, I’m just stressed with work. But the truth is I just really don’t want to be their friend or talk to them outside of what’s required. I’m perfectly civil when talking about work related stuff IMO. But I do worry that, since I’m more friendly with the others in my group, this will be seen as me being hostile towards this one person by my PI or whoever.

So just wondering how other people have handled similar situations 😬 I know there will always be people at work that you don’t get along with, but I’m just not great with interpersonal stuff.

Does anyone have experience with the new-ish NEB Q5-XT polymerase?

I do a lot of longer amplicon PCR (∼10kb) for cloning and genomic integration casettes and generally my go-to has been SuperFi II but always looking for better and cheaper alternatives. I have used regular Q5 and have found in general I get lower yields, particularly for larger amplicons, and it takes significantly longer.

and my failed y2h

Something I've been dealing with recently that I'd like some insight on:

My lab has recently bought an autoclave and before I took over, we had bought many fume hoods. We own these entirely and the vendors for both are withholding the admin credentials. I could possibly force the issue, but haven't as on a day to day it isn't an issue.

I'm not interested in why, I'm just interested in what the hell is going on? How is this any different than buying a car and having the keys kept from you?

Hi all,

When I have long experiments to run I like to listen to videos or podcasts on my headphones (I'm sure I'm not the only one !). One issue I got myself into is that I find that most of the podcasts out there are just... kinda boring ? They are aimed at a large audience so will usually stay on the surface level and are not that intellectually stimulating. Take for example the ologies podcast : you have experts in their respective fields but the host only asks basic questions and doesn't let them actually say anything of depth, kind of frustrating.

I'm looking for stuff that delves deep and that doesn't take its audience for dummies ! I want to hear experts yap about their interest for hours, stuff like that. I've found that a couple of the Big Think interviews were good, I also like the style of Tor's cabinet, which is really well researched.

Let me know you favourites !

Hi everyone! I've been having some problems with my cells being contaminated, so I wanted to check whether my media (DMEM/F12 + GlutaMAX) could be a source of contamination. I put 2 mL of media in each well of a 6-well plate and put it in the incubator. Two days later, I checked, and this is what I saw. I'm not sure what this implies about my media, since there are no cells or any organic material in the wells.

I’m a first-year PhD student in immunology (wet lab, not computational), and I’m trying to figure out my next rotation and, eventually, my thesis lab.

Option 1: A brand-new lab. The PI just started, but he trained at the same institute, so he already knows his way around the university and has connections here. I’d be his first PhD student. I genuinely love the research.

Option 2: An established lab. It has a track record, senior students, but I’m less interested in the research.

My main worry is time to degree. I really want to graduate in five years, and I’m scared that joining a new lab will slow me down with setup, mouse colonies, approvals, and no senior lab members to learn from. At the same time, I know five years is a long time to spend on research I’m only lukewarm about.

For those who joined a new lab as the first student, or chose between options like these:

• Did being first in a new lab delay your graduation?
• What red or green flags should I look for during the rotation?
• What should I ask the PI before committing?
• Did anyone pick the “safer” lab and regret it, or pick passion and regret it?

Any advice is appreciated!

I got my PhD a year ago, with a paper ready to be submitted. We got reviewer comments back recently that prompted me to look at some data more closely. I'm realizing that there may be a huge issue that affects ~70% of my PhD experiments. (Basically, that an animal stock I was using may have been mixed up or contaminated). This could render several of my conclusions invalid. I have to tell my advisor, of course, but I am freaking the fuck out. Any words of wisdom would be greatly appreciated.

EDIT: my work was with flies. I am concerned that a fly stock that I was using for a lot of my experiments was mixed up with a similar stock, but lacking a crucial genetic element that would affect the interpretation of the results.

Whenever we spin down insect cells (ExpiSF) infected with baculovirus we get a multilayered pellet. A lighter whitish-brown layer on top, a brown layer on the bottom.

The lighter layer is tight enough that it stays intact when pouring off the media.

Has anyone else seen multilayered pellets? If so what are the top/bottom layers?

We typically harvest at 80% viability post viral infection as measured by a cell counter. This is typically 48-72 hours for us.

I get inconsistent answers from AI tools and Google search so wanted to ask here.

Cells are infected with baculovirus. Appear to be free of other contamination under the microscope.

AI tools say that the top layer is induced/expressing and the bottom layer is uninduced/low expressing but that seems suspect.

I am doing my PhD in an institute and I mostly fear to go to lab because of my lab seniors. Right now, I need to work in the lab and I am feeling really insecure to start my work. They had already mocked me for working differently. I have learned certain techniques during my college days and that's how I have been working in this lab. But, my seniors work in a different way, their handling is completely different. So, they have mostly judged me for working differently. Right now, I am scared to even ask for the required reagents from them to start my wet lab work.

Hello, I have extracted RNA from the hippocampus and I need to run cDNA synthesis(iSCRIPT BioRad) and qPCR(iTaq universal Sybr green). I have 12 samples and 4 genes including housekeeping gene. I have 2 questions:
1) how much RNA should I add to do the cDNA synthesis?
2) how much cDNA and primers should I add for the qPCR step?
I’m trying it for the first time and no one in my lab knows how to do it. Also, if there are any tips/ suggestions I should do, pls lmk!
Thank you so much

Hey everyone,

I'm a PhD student at a state university in Idaho. My proposed research (I am first-year and about two months in so I don't have the details of the project lined up yet) will investigate ligand-directed GPCR trafficking. Most of the tools I was planning to use were designed using the HEK293 cell system (i.e. BRET G-protein/beta-arrestin assays).

People in my department (including my PI and other PIs) have been repeating the notion that we are not allowed to use HEK293 cells because they are derived from human embryonic tissue (Idaho is a very pro-life state). This throws a wrench in my plans. However, I can't find a specific rule at our institution that forbids use of the HEK293 cell line. My understanding is that many many generations ago, this cell line might've been derived from human embryonic tissue, but when you get cells from ATCC for example, the cells are basically "clones" of that original line, so technically embryonic tissue wasn't harvested again after that first isolation of the cells.

I'm a bit puzzled. I'm also not sure how to work around this barrier. We could use CHO cells or I could get trained culturing rodent neurons (I have some basic training, but I was never successful).

I am doing RNAscope stainings on FFPE tissue using the manual Multiplex Fluorescent v2 kit and I often get these weird structures in my stainings, even in my negative controls (i.e. no probe, only probe diluent), see attached image. They glow in all channels and I can't figure out how to get rid of them because somehow nobody on the internet has ever posted a similar image. Does anyone here have an idea what these things are and how I can avoid them? Thanks a lot in advance!

Hi everyone! I had an in person interview last week (Monday) that went super super well and was given a start date (estimated) and was told they would love to have me on the team. Afterwards I emailed thanking them for their time and they emailed me the estimated salary range. I replied back stating that it was in my expectations and would be happy to sign an offer letter whenever it is convenient for their team. It has been a week since I sent that email and I haven’t heard back. I would really like written confirmation of a job in order to plan my move across the country lol. When do you all think it’s appropriate to send a follow up requesting a more concrete timeline? Thanks!!

For context this is a job with the gov working in a core facility.

I’ve been struggling to do basic quality checks of RNA on agarose. I tried a bunch of times using agarose and a GreenSafe dye and saw nothing at all for RNA. It just never showed, not even degraded. I contacted the company who made the dye and they said RNA was hit or miss and the dye was really meant for DNA.

So we purchased some SYBR gold, and this is a run using that. 1% tae agarose, no denaturing. On the left is DNA ladder, 10kb. Then the two RNA samples extracted from whole blood using a Zymo silica column kit. Then four DNA samples using a Separate Zymo kit. The RNA kit used on column DNase I.

The DNA looks mostly fine I think, although there is more smearing than I’m used to seeing on GreenSafe. But the Gold is way more sensitive so maybe that’s normal?

But the RNA I can’t tell what’s going on. I don’t see any peaks, but it’s also not clustering at the bottom either like pure degradation would give. I redid the RNA again after this and tried various other smaller loads in the lanes and it mostly looked the same.

Is the RNA degraded? Is this DNA contamination? Is this due to not using a denaturing gel? I measured on qubit and it was about 20ng/uL, and these lanes had about 200ng in them. Sorry for the image quality this was just shot using a phone. I really want to have some repeatable quality check before moving onto PCR but I can’t seem to get the basic agarose working for RNA. Any guidance would be appreciated.

I finished my master’s recently and now work in research, and I’m still figuring out how to keep everything straight. For those of you doing research, what tends to get messy for you—keeping track of papers, notes, experiments, deadlines, or something else?

We have this nutrition-related project at our company that is partnering with a university.

This project was originally proposed last year June, and I was unfortunately one of those young employees who wanted to be proactive and prove myself. Now I sorely regret it.

The project has been rocky from the beginning. The person who proposed it used wrong ingredient ratios, resulting in three reformulations, so I had to re-do the INFOGEST three times. INFOGEST is such a tedious experiment and can get expensive because of salivary amylase.

That coworker got laid off early this month, and another employee who has been on a long term sick leave and now is back on a half time basis, took over.

I have to perform INFOGEST for like, 250mL of 12 samples. But then I have to take close to 100 aliquots of it.

What I dont 'get it why I am being told to do all the lab works....I am not even an INFOGEST expert and my job isn't even related to nutrition. Is this normal?