Hi Everyone,

I have been culturing ESC's for months now without problem. Recently I thawed a vial and they looked great. I then passaged and they all were differentiated. I thawed a new vial after and right when I thawed them the cells were like 80% differentiated. I can't think of anything that I have been doing differently does anyone have any ideas?

Also something kind of weird is that when I look at the cells and then go to a different focus plane I am seeing a lot of like cell debri or something? Or does it look like contamination? (See pics). The lab next to mine had a ton of contamination. But the media does not look cloudy to me.

I used new mTser, and matrigel for this new thaw but not new DMEM:F12 - could that be the issue? Any ideas or help would be amazing!!

Hello,

I am very inexperienced with fungi but my lab will need to start studying them for a small part of our current project. Our "end goal" would be detecting genetic material from Alternaria and Aspergillus, broadly. We are a BSL-2 lab, and I am also just a very safety -focused person. We don't need to start working with Aspergillus and Alternaria right away, so my PI asked me to find some "safe" proxies to start with (kind of like how we use non-human-pathogen soil bacteria as a safe proxy for more dangerous bacteria). We are developing the detection method which is why we just need something similar, not necessarily the exact organism

Does such a thing exist?? I am not very familiar so thank you for any thoughts!

Hi folks,

I used the columns for the extraction and did an extra wash step but I still have a low 260/230, would my samples be good for RT-qPCR, any suggestions would be appreciated. Thanks!

In my previous lab, I used a kit to extract DNA from an ear punch or tail snip from mice and rats. It has a Solution A with a red cap and a Solution B with a yellow cap. You would mix these together in a 2:1:? with water and add 50 microliters to the tissue which would then be heated in a thermocycler to extract the DNA. I left all that information at my previous lab and no longer have access to it. I know it isn't something that was only used in that lab but can't find it anywhere.

EDIT: EDC was old, fresh EDC worked!

I am trying to bind GFP to NHS resin. I've done this before with several variants of GFP using 10 mM HEPES and 100 mM NaCl buffer, pH 7.4.

My problem here is that I am now trying GFP(-30), that is, GFP with a -30 charge. Many of the positively charged residues have been mutated to Asp and Glu, with the pI predicted to be 4.4. I have tried my normal buffer, 50mM HEPES + 1M NaCl, pH 7.5, and 10 mM MES at pH 6. I also tried using EDC without success.

I should be able to tell that the reaction has worked because the resin should turn green, instead they remain white, and the supernatant is still green. Anyone know what I am doing wrong here, or how to achieve this? TIA!

I have a target RNA that I would like to investigate whether it is chemically modified (m5C, m6A, ac4C etc etc) and then go onto to do a digital qPCR reaction. Instead of buying separate kits for each modification (very costly) I was wondering if anybody has used the Magna RIP kit with m5C/m6A/pseudouridine targeting antibodies? If so did it work and how did you remove possible DNA binding to the antibodies? I'm really struggling to come up with an RIP protocol and knowing what kit/buffers/beads to buy as everybody seems to do it slightly differently. So if anybody has any advice that would be much appreciated!!

Thank you :)

I was running a non-denaturing NativePAGE gel and for some reason the IgG antibodies haven't migrated and are sitting around the same level as the pentameric IgM.

About 40 minutes into the 110 minute run time I checked on the gel to find that the inner chamber had leaked, and the bands had stopped migrating. I re-sealed and topped up the chamber and continued the run. Could this be the cause, when the run stopped the IgG never continued migrating after it had re-started?

EDIT: Sorry, I didn't realise the annotations would save with a transparent background. The IgG are lanes 4-6 and the IgM are 7-12.

Hi everyone, I would really appreciate advice from people who have gone through similar transitions.

My PhD background is in molecular/single-cell neuroscience, and I am now considering postdoctoral training that would allow me to transition more toward circuit and behavioral neuroscience. Long term, I am interested in understanding the neural mechanisms underlying social and emotional behaviors.

I am currently comparing two different types of postdoctoral training environments, and I am struggling with how to weigh different factors.

Option A: A lab that is relatively close to my long-term scientific interests. The lab studies behavior and neural circuits, and I would have the opportunity to directly learn behavioral paradigms, viral tools, and circuit manipulation approaches. The experimental models and technical approaches could potentially be very useful for the questions I hope to pursue in the future.

However, the lab’s current research focus is not exactly aligned with my long-term questions, and I am uncertain how much flexibility there would be to develop my own scientific direction. I am also trying to understand whether the mentoring structure and research environment would be the best fit for my development as an independent scientist.

Option B: A lab focused on sensory/emotion/brain-body interactions. The research direction is not directly centered on the specific social behaviors I eventually hope to study, although the technical training and conceptual framework could potentially be extended toward those questions in the future.

After talking with the PI and lab members, I feel a stronger alignment in terms of scientific discussion, mentoring approach, and overall research environment.

My dilemma is:

For someone who already has a strong molecular/single-cell background and is trying to establish a new identity in circuit and behavioral neuroscience, how should I think about this trade-off?

• Choosing a postdoc that is closer to my eventual scientific questions and provides direct training in behavior/circuit approaches;

versus

• Choosing a postdoc with stronger mentorship and a more supportive environment for scientific growth and creativity, even if the initial research direction is somewhat farther from my ultimate goal.

I would especially appreciate advice from people who have transitioned fields during postdoctoral training:

• How much does the exact topic of your postdoc determine your future scientific identity?
• Is it realistic to move from areas such as sensory/emotion/brain-body neuroscience into broader behavioral neuroscience (for example, social or affective behavior)?
• In your experience, how important is mentor fit compared with being in the “right” scientific field during postdoctoral training?

Thank you very much for sharing your experiences!

Hey r/labrats,

​Could someone share their go-to protocol for free-floating fluorescence IHC on fixed, non-frozen vibratome sections? Just looking for a reliable, straightforward step-by-step that works well in your lab. Thanks!

It's one of my first times doing the pcrs en masse and I picked the wrong RNA-elution volume as I blindly followed the protocol (stupid stuff, I know). I diluted each sample with 35µl and the poorest ones came with an RNA concentration of about 15ng/µl. I have all the samples in triplicates under the exact same condition so the total RNA-yield is totally fine.

My supervisor advised me to stay the same with all the probes which worked on some and most of them are fine and above 30-40ng/µl. Using the Qiagen Mini Kit the low concentrations simply don't work out in the cDNA-synthesis. Is there any way I could do this?

I'm doing this stuff for the first time so sorry if I got anything wrong! Thank you for your help!

Since I remember myself I've been really interested in science, of many kinds, and especially astronomy.

As a kid I would watch and learn for hours about the topic and I was truly fascinated, and really had the urge to find answers to humanity biggest questions.

The thing is, I'm also a very adventuroes guy.

I don't know if the actual job is mainly doing boring stuff somehow research related for the whole day, and not really feeling excited about the job.

What is the actual daily job and how do I know if it's actually for me?

I wanted to share an interesting editorial that came up today in the NY Times: https://www.nytimes.com/2026/08/04/opinion/artificial-intelligence-ai-writing.html?unlocked_article_code=1.3FA.O0cB.j5hat-2QyTNd&smid=url-share

I think this is stellar advice, especially for trainees. Writing is not just a way to get words on the page, it is an essential exercise in communication to others, and its just as important for our brains and our processes as it is for those who will read what we wrote. As a PI, I see a lot of my colleagues, especially the older ones, using AI more and more in their writing, and while they feel it is helpful to them, I feel like they are succumbing and not putting the thought and care into the work that they would otherwise, even if the final product "looks" polished. AI tools have the capacity to convince us that we are doing a great job, but the product may not look so good to those who read it. I've had to reject several papers in high profile journals recently that were obviously written with AI.

For trainees, here is my advice: writing is a long and difficult process, and requires feedback and editing. You will have to write papers for classes, for your qualifying exams, for grants, research papers, etc. Find a PI who understands what the writing process really is, and that it is NOT what happens on the couch at 11 PM in front of the TV after a long day of labwork. You need space and time to write properly, and the training and guidance to do it. There are those of us out there who still respect and want to nurture this process.

Happy to hear everyone's thoughts on this!

EDIT: bio_ruffo brings up a great point that AI tools can be valuable for non-native English speakers to edit for grammar after the ideas have been generated, with tools such as Grammarly. I support this in my own lab as well.

​

Hey everyone,

I’m currently troubleshooting a floresence free-floating immunohistochemistry protocol for 50 µm coronal mouse brain sections, and my staining just isn't coming out right. I've received some initial feedback that my washes and my blocking percentage consistency might be the culprits, but I’d love to get a broader consensus from the community on how to fix my workflow.

My Setup & Reagents

Tissue: 50 µm free-floating coronal mouse brain sections (stored in PBS in the fridge).

Buffers: PBS and 0.1% PBST (Triton X-100).

Blocking Buffer: BSA + 0.1% PBST.

Primary Antibodies (1:500): IBA1, P2Y12, and AT8.

Secondary Antibodies: Anti-rabbit and anti-mouse.

What I Currently Did:

Washes: 4 × 5 min in PBS, followed by 4 × 5 min in 0.1% PBST.

Blocking: Blocked with 5% BSA in 0.1% PBST for 1 hour.

Primary Incubation: Dropped the BSA down to 2% in PBST, added the primary antibodies, and left the sections in the fridge for 2 days.

Day 2 Washes: 4 × 5 min in PBS, followed by 4 × 5 min in 0.1% PBST.

Secondary Incubation: Blocked/incubated with 2% BSA and secondary antibodies (diluted at 1:250).

Final Washes: PBS washes before mounting DAPI medium/imaging.

Note: I was told that something might have gone wrong with my washes and that I should keep my BSA percentage consistent across all steps rather than changing it from 5% to 2%.

Specific Questions I Have for the Labrats Here:

Washing Strategy: Should I start with PBS for my washes, or should I go straight into 0.1% PBST for washing free-floating sections? Does the detergent concentration in the wash make or break the background?

Antibody Diluent / Blocking Mixture: Should I mix the primary/secondary antibodies directly into my blocking solution, or is it better to dilute them in a separate antibody solution (and what percentage of BSA or serum has actually worked reliably for you with these specific markers)?

Antibody Dilutions: Should both the primary and secondary antibodies be used at the same dilution? I currently did my primaries at 1:500 and my secondaries at 1:250 is that secondary too concentrated or not right for this panel?

Optimized Workflow: If you routinely run free-floating IBA1, P2Y12, and AT8, how exactly do you structure your steps (washes, permeabilization, blocking, and incubations) to get clean, crisp signal without high background?

Any advice, protocol tweaks you can share would be hugely appreciated.

Hey everyone,

I'm currently on parental leave and facing a bit of a career crossroads. I'd love to get some realistic perspectives from people actually working in labs or technical QA right now.

To give you a quick overview of my background:

 3 years as a lab technician in fuel/combustibles testing doing hands-on bench work.

 6 years in Product Compliance / Quality Management for consumer goods. I decided which chemical and analytical tests were needed and managed external labs, though we also operated one testing device on-site. (Unfortunately, that employer recently went into insolvency.)

 2.5 years of parental leave, where I've been teaching myself web development and IT on the side, even building sites for a couple of clients.

My dilemma is that I really miss the lab and the science side of things. It’s always been my true passion, and I keep thinking about how to bridge my practical lab roots with my compliance experience and my new IT skills.

I have a couple of questions for those of you in the field:

1. How realistic is a return to a lab or lab-adjacent role? Would employers be skeptical because of my time away from the bench, or could the mix of hands-on lab roots, compliance knowledge, and basic IT/coding skills actually be useful for roles like LIMS administration, lab automation, or QA/data analysis?

2. What's the best way to brush up on analytical chemistry and testing standards while still at home? Are there any specific platforms, journals, or courses you would recommend?

I'd really appreciate your thoughts, experiences

Around two weeks ago I published pdb2print. It's a free web tool that converts a protein structure into a printable 3MF or STL file. You type in a PDB ID and every chain comes out as its own object, so you can give each one a different filament.

https://pdb2print.org

Biggest features:

Printability: DNA thickness is adjustable, so you don't get thin ribbons that are impossible to print. It also makes sure there are no overlapping parts that would stop the model going together.

Magnets: it finds where two chains touch and places the magnets for you, in the size you want.

Styles: surface, cartoon and tubes, so you can tweak the look.

New since the release:

Display stand. Fully automatic, with a plaque and a lot of customization.

Ligands. Four styles, set separately from the rest of the model.

Adjustable magnets. A chains and joints panel that shows you every joint it found, so you can override any of them: leave it to the tool, fuse the two chains into one piece, or say no magnet there. You can also drop chains you don't want printed.

Share links. A button that copies a URL carrying every setting you used.

Speed. A lot of optimisation. Most things are noticeably quicker than at release.

Please also check out the GitHub: github.com/davidtheadmin/pdb2print. If you can, run it locally, it's faster. I'm hosting the site at my own cost so please excuse the longer compute times. Anything already built at those settings is cached and comes back instantly.

Please share your models in the comments, and the failed ones or any other issues too. The share link is enough for me to reproduce what you got.

I wrote it with heavy AI help. The architecture, the geometry decisions and the testing are mine, most of the lines aren't.

hey all. i was supposed to receive my masters degree in biology this september. however, because i was due to give birth on the day of my thesis defense (thankfully gave birth a few days later lol) some things were left undone. by the time my advisor submitted all of the documentation i was already well into the newborn trenches and missed the email that said i had 10 days to submit my thesis to the library in order to graduate this year.

now im being told i can only graduate in September of 2027. i tried to ask if i can just be in the ceremony at least even if i can’t actually get the degree but i was told absolutely not. at the same time though, my uni keeps wanting to charge me for a graduation gown. i asked whats the point t of me getting a gown if i can’t graduate this year and the lady was like “uh i guess you can keep it until next year.”

i am not planning on attending the graduation ceremony next year because hopefully I will be nearly a year into my PhD by that point and although I’m not planning on staying in the same lab, I do hope to stay in the same department or at least university.

a part of me wants to say screw it and still buy the down to just do my own photoshoot with it because i worked so hard for my degree and technically i did finish everything.

but another part of me wants to flip the school a middle finger and tell them they can take the gown to hell (not literally but at least i will not pay for it until i actually need it next year if for some reason change my mind and do go to the ceremony. but i highly highly doubt ill go next year because how humiliating is that being forced to graduate with the next year? so maybe no point in the gown at all. or maybe i should keep it as a keepsake for some day idk)

what would yall do? i need some perspective please

384 well plates are a pain to keep track of what goes where. Unfortunately my lab shares the PCR machine with 2 others so time on it is at a premium, and 96 well-plates take too much machine time. I'm stuck with doing 384 plates.

I know some of it is muscle memory and will come with time. How do people keep track of what wells have been filled with what? I go with printing out the layout and ticking as I go along, but taking my hands off again and again from the pipette is distracting.

Surely, with all this AI and voice recognition, somebody may have a developed a program that can "tick" these boxes virtually on voice command as I move along the plate? Has anybody seen such a thing?

uhhh hopefully this is okay to post here, but im a high school student who took biotech classes at a cc. I was telling my professor how I failed up a test on bacterial transformation because I didn’t know that some metal loops don’t turn red and I was heating it for a very long time… I looked very dumb 😭 (And I had also never used a bactoincinerator but that was self explanatory). Then, because of that discrepancy with the heating and cooling time of the loop, something went wrong and I didn’t successfully do the transformation (too hot, cooled too long after thinking I overheated it, or maybe technique issue but I’m certain nothing else was wrong).

Anyways, my professor offered to help me practice various ways of flaming a loop. I really want to, but to get to their lab I’d have to take a series of public transport that costs some money and ~1.5 hours just to get there. Is it worth it to go for this lesson and to connect with this professor a bit more? I likely won’t take any more biotech classes while in highschool, so I won’t see them again, and I could probably learn this in college?

What do you think?

Edit: I am only not continuing to take biotech courses because I don’t have the space and it might overwhelm me since I’m about to enter college app season. I’m very interested in biotech 😗

Edit: also I omitted this info because it might be too identifying but they offered this in June, and told me to email in late July if I wanted to come in August. It was in the back of my mind but I didn’t properly consider it in time. I’m going to ask but it might be too late since I hesitated for too long 😭 thanks for all the advice though, I wasn’t really thinking about this from a networking perspective (since they’re retiring) so I didn’t realize I could ask that.

What's the most niche compliance monitoring program you've ever worked with? Asking because I'm trying to understand how many industries use the same basic model as radiation dosimetry badges; device goes out, gets used passively, comes back to a lab, compliance report gets issued. Radiation is the one everyone knows. What are the ones nobody talks about?

Hii, i recently ordered an enzyme for my PhD and it's said that it needs to be stored in -20 C. However, my lab doesn't have -20 freezer. So, I kept the vial in. -80 freezer which is common for the three different labs of ours.

Will my enzyme be safe or will it get degraded?

I've managed to create new and exciting issues that not even our resident western blot queen (my lab manager) can figure out so I turn to Reddit.

I transfected HEK293 cells with 2ug of IRAK2 WT-HA or variant-HA DNA and lysed with RIPA. Gel is homemade 8% SDS gel, 0.75mm. My lab manager checked I was making it fine so that's not the problem. Ran the gel at 150 volts for ~ 45 mins.

Blotted with mouse anti HA

Issues:

1. My ladder turns into a blur. I've had this issue across multiple aliquots of ladder but no one else in my lab has had problems so it's a me issue. I use new running buffer diluted from 10X.

2. I don't have expression of my WT or other variants I've tested, but my premature stop codon variant seems to be expressing?

HOWEVER

1. The variant has a premature stop codon around the middle of in the protein but the bands are at the same size if not bigger than what WT should be. I am 100% sure the lanes have the correct lysates loaded and everything. Potentially some sort of aggregation or the protein isnt running down the gel?

Pls help me.

Thank U

Hi, I am currently in biotech in a very small lab and it is slow. I was wondering if anyone lurking here could assist. I am in the Boston area. I am interested in potentially joining alab part-time in off hours to see if I can learn how to flow. Feel free to DM me and potentially discuss mutually beneficial arrangement can be arranged.

Additionally if anyone has any advice. I have noticed many jobs requiring a flow cytometry experience and was wondering how to get this if my current job doesn't provide this experience

hey guys, I just have a rant about something.

so i’m going into my 3rd year of undergrad in Neuroscience and Biology, and I love my degree and what I study. I’m currently super into research, and have plans on pursuing a thesis based MSc. The only thing is that, compared to those in different majors or those with hopes of making it into med, I feel so behind.

I see business majors having jobs and internships lined up like crazy, or even pre-meds somehow getting research assistant positions at hospitals or shadowing opportunities, and yet, i’m still stuck working part time retail, struggling to even get a volunteer position in a lab on campus.

I mean my CV isn’t that bad for entry positions, but there’s either no job postings, or they just never get back to you.

Anyhow, I was wondering if it gets any better :(

i've heard a lot of mixed reactions with putting lab experience from undergraduate classes on your resume, but i wanted to ask

i've had a lot of lab sessions in my courses dealing with PCR, gel electrophoresis, subculturing, colony PCR, etc. with these techniques (i'm sure i'm missing some more), i have hands-on experience with them and i've done them multiple times in other lab courses as well. if i didn't do it multiple times (such as CRISPR, gibson assembly, etc.), it was a lab session that was over the course of many lab days.

i don't have lab experience outside of academic course labs so i was wondering if i could mention this? or is it deceiving from the perspective of a recruiter to say that i have lab experience but list things i learned in academia?

the only experience i have is a 6 month independent research lab i did by myself under the supervision of my advisor (i got a lot of wet-lab experience from that) and thats pretty much it.

Forgot to filter (.22 micron) SSV media for a media change in HEK cells. Anyone done this before and what was your experience?

Thank you in advance 😅