What is this and how do I fix it? I grew fibroblasts from skin punch biopsies and froze samples after the 3rd passage. I am just getting back to them, and two days after thawing and incubating I get a sheet of these cells. I cannot just replace these samples and I’m terrified of losing the line. Any insights?

Also, the dark spot in the middle is just a lens artifact.

From - A badly dehydrated 2nd year PhD student

Hi,

For anyone who won a postdoctoral fellowship from the ADA and is receiving their postdoc salary from that, do you get the entire amount that ADA sends your institute or does your institution pays you salary at the NIH level and keeps the rest for fringe? ADA fellowship is pays more than the NIH minimum and according to ADA’s policies the institute is not supposed to keep any amount of the salary for fringe. But I have heard from a few of the fellows that is happening at their universities. Is this normal?

TIA!

I have been stalking this thread for a while now, and myself drafted and deleted a couple of posts on the topic I struggle with.

This last whole summer I worked in a lab as a full-time undergraduate researcher under one of the post-docs. At the beginning of my job I disclosed with my supervisor that I struggle with physical disability that often times causes flares of chronic pain and leads to cognition issues. In short: pain = trouble focusing.

At the beginning we built a decent rapport, and work went great. After a while I noticed sharp, frustrated reactions to my clarifying questions and overall lack of understanding that I am not on the same level as them. I brushed it off as the curse of knowledge bias. It has gotten worse, and I reached the wall in our communication. I am stressed to go into the lab, scared of asking questions. This leads to stupid mistakes, which in turn can lead to failing the experiments.

I take ownership of my mistakes, and take notes to make myself a better scientist. However, today my supervisor made an extremely unprofessional and out-of-pocket remark that made me question whether I can continue to work with them. This is on top of often suggesting that I do not think. What should I do now, talk to the PI or just leave?

Hi all,
do you have protocols or electroporating parameters to successfully deliver two different plasmids (8.5 kb each) in HepG2 cells?

The parameters that I selected as optimal when trying with a simple GFP construct seem not to work with the two plasmids, but the overall amount of DNA is the same. Maybe they are too big?
I’m using the neon xt from thermo

Thanks!

EOL-1 cells in RPMI1640 w/ 2mM L-Glutamine supplemented with 10% FBS and 1% P/S

Hey all! I’m hoping to get some inspiration on decorating my bench space to make it more cozy. I work in a lab where students do not have any office space so we are assigned a bench space instead. It’s an ergonomic nightmare but oh well. What kind of things have you added to your space to make it feel more like yours? I should note it is a BSL2 lab which does limit my choices a bit.

In the paper from Silvana Konermann and Feng Zhang about dCas9-SAM for CRISPR activation (Konermann S et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature, 2015 Jan 29;517(7536):583-8), a list of guide RNAs for each gene is provided.

Just curious here - has anyone tried any of the guides from the list to see if they work? I know they were computationally derived... I want to purchase a construct with a guide from this list and dCa9-SAM, but it is not cheap, so I'm wondering what people's experience has been in general with using guides from this list. Any insight would be most appreciated :)

Cheers!

Has anyone here switched postdoc labs? What was your experience like? Did you tell your supervisor beforehand? Did you completely change institutions? What was your strategy? I am pretty sure I am not getting the experience I need in this postdoc-- pretty sure the project is a dud (but PI loves it), PI is a micromanager (walks by my gels and comments on the amount of loaded), doesn't allow postdocs to take any of their projects to faculty positions (could be why getting faculty positions seems rare in this lab), I'm not learning many of the techniques I joined the lab to learn and am not being allowed to, I am not being allowed to maintain the techniques in which I gained expertise during my PhD (which was a condition of me taking this offer, so feels like a bait and switch). I just don't see myself progressing here and the environment is kind of meh scientifically (no one talks to each other about science, no paper discussions, lots of people who just talk to ChatGPT to plan their research instead of colleagues)... I've been here a few months but I know I want out. I am not deadset on being a PI, so applying to industry is an option, but I really enjoyed my PhD so I think a good fit postdoc lab could be enjoyable for me.

I'm in my 7th year of my PhD. I say 6 years and 6 months. No publication at all. Desperately trying to publish something. Made a barely holding together bioinformatics paper and is getting rejected everywhere. Apparently the topic is too niche or doesn't suit any journal. I do not know why I am posting this here. Now I'm trying to publish a protocol in Bioprotocol. *fingers crossed* But that doesn't cut it because I need two Q1 journal article to get the degree. At this point I'm hopeless and I can't write even one sentence for my thesis. Feel like I'm in this longest downfall of life and the end is not near. At this point I can see a mental depression lurking around the corner of my mind.

I used to write, draw and take photos and teach pupils and had a nice little life before PhD

Now it feels all grey. I do not do anything other than paper work and writing and yet nothing to show. my peers have either completed or in the process of submission. Above all, my stipend is over it's been 2 years I am surviving out of goodness of good old friends (who never choose phd and survived in industry).Barely surving with a credit card debt and personal debt. I'm in my early 30s and still sometime i have ask rent money from my retired parents.

Does any of this pays off at the end? or does it ends at least?

context: From India, no option to get a masters degree. My Bioinfo work is a statistical model of a particular cancer. Paper work might be a Indian academic term means just doing the editing/rewriting/fixing/expanding since its getting rejected.

Hey, recently I have seen a lot of people asking biotech companies,chemicals companies,etc. for merch (Eppendorf,...). Well, I tried and no replies :)))))) Just a student who wanted to go to Biomedical Science so may as well get some luck (yes I know luck is not the only thing that matters, so I study a lot too hehe). Have you guys got merch from any of them?

Hello, I do not have much experience running gel and I have been trying to optimize on a ~20cm electrode distance gel casting device. This is my second time running the gel and would like some advice. It looks sort of slanted??? at least more than I expected on the first few wells on the very left. If you have any tips Id love to know. I provided the photo below.

I have been checking the gel every 15-20minutes and determined that 1hr and 45 minutes may be good enough to see the whole ladder, though based on my photo I might run it longer at 2+hrs to get better seperation on the larger bp ladders

Specifications:

6x Tri DNA dye at 1:10 ratio.

I ran the gel for about 120V for about 1hr and 45 minutes.

For the buffer, I am using 100mL of 1x TAE buffer + 1% agarose (1gram). For the Ladder, I am using 1kb plus lab ready Thermo ladder. For the dye, I am using 10000x SYBR Safe diluted to 1x. I use of 10uL of SYBER safe for pre-staining.

The liquid and the gel is not overheating. I load about 10uL of the lab ready Thermo Ladder and the same volume for all my DNA samples.

Thanks in advance.

Hi, I have just started this new job and there had been no specific mention of how long they wanted me to be there. I am a recent college graduate and decided to get a job instead of going straight into a grad program. However ultimately, I would like to go to grad school and get a PhD. My appointment is one year, which I can renew. Therefore, my plan was to do this job for at least a year and start applying to grad programs, even if I decided to delay starting the program till Winter of 2027.

However, I talked to the lab tech who was in my position and leaving and she mentioned that she would be training my till early December (then she leaves) and I was thinking that I would probably be the next person training the next lab tech. She also mentioned they hired a lab tech before that left after a year and she stayed because she and the lab manager worked really hard to run and create this lab (it is legit a two person lab, run by a single person). She is therefore trying to find a good replacement for her. I am feeling worried that maybe they anticipated I would be here for longer than I was actually prepared for, so I’m not sure what I should do with this information.

Does anyone have any advice on how I should proceed?

Hello! I am currently in the middle (nearing end) of my PhD, where my project focuses on Notch1 in HER2+ breast cancer. A big part of my project is verifying the expression differences under various conditions, all in vitro. I have been able to successfully and repeatedly get results for immunofluorescent staining, which has been great.

Two things that I have been doing over and over and over (different samples, protocols, etc.) is western blotting and flow cytometry. I have not had a single band show up for Notch1 in either my HER2+ cell lines or control (MCF10As). In flow, my cell lines basically look like they do not stain, as my last results showed SKBR3s containing no HER2 at all.

At a recent conference, I spoke to two separate researchers where their labs also worked with Notch1, and they had the exact same issues where IF staining worked, but flow and westerns did not. One lab completely gave up on Notch and the other is just going off of IF staining.

Has anyone else had a similar issue to this (not necessarily just in Notch1)? I am trying to research other assays to complete (I have an ELISA kit to try eventually), but it seems like I am quickly running out of options. I am fine with writing that Notch1 is not a HER2+ BC biomarker, I just need some resources to back this up and to help show I'm not just an incompetent researcher lol. Any advice, papers, anecdotes, or anything at all would be a great help, thank you!

I work in a lab that handles a lot of aqueous corrosives, including fluoride, and a diaphragm pump seems like a sensible choice for what we need, which is vacuum filtration and similar simple tasks, mostly on the 100-300ml scale.

Does anybody have any brand or product recs? I don't trust any of the lists I've found online, as either they give me AI vibes or they only list Amazon-available items. Although given how low-power this application is, if the answer is indeed "Amazon is fine" then I can work with that.

Edit: it looks like the Amazon special is indeed a reasonable place to start. There's a Rocker 300c for $450 that appears to be... Good enough , from a vacuum and corrosion resistance perspective.

Thanks for your input everyone!

I had gene which was codon synthesised and cloned by manufacturer in puc plasmid
Which I subcloned in pET vector and transformed in dh5 and saw the fallout at the correct size upon double digestion check. I followed it by transformation in arctic ecoli cells( induction for 24 hrs at 12 degrees C using 1mM IPTG) . I did a small pilot clone check by doing a western blot using anti his ( protein has a c terminal 8x his) and I detected the protein at the expected size and compared it with a positive control as well.
However after doing purification with Ni nta beads (overnight 4 degrees using batch method 500ml culture) I am not getting any protein at all!
What could be going wrong? Any suggestions?

If your work depends on grants, you should review the proposed NIG policy changes, and submit comments.

Late Friday night, the NIH posted a proposal for peer
review changes: "Request for Information (RFI) on Proposed Changes to Reporting Outcomes from NIH
Peer Review". This change would not report the impact score of a peer review grant to researchers and the NIH.

Impact scores give not only researchers an idea of how well they performed, but also guidance on how close their grant may be to funding to re-strategize.

If this will affect you in any way. I encourage you to independent read into the proposed policy changes, and submit comments.

Say who you are and why this matters to you, taxpayer, patient, family member of someone affected by NIH-funded research, and explain in your own words why you think grant scores should stay transparent. Real, specific comments carry more weight than a form letter.

The deadline for comments in October 13, 2026.

Measured my yields from some gel extractions earlier and one of my samples was only 6-7 ng/uL, with a decent A260/A280 (~1.8) but a terrible A260/A230 (~0.03). Can I be confident that there is ANY DNA in this sample whatsoever, or is it just pure background noise?

Just wondering cause I only need a couple ng for downstream applications and it would be a real pain to have to remake this sample in particular

Howdy everyone. I am not good at math because i get confused and feel like I am going crazy with it/thought about it for so long it feels incorrect, so I’ve come to ask for help.

I am diluting a stock sample to inject 10uL of samples at the following concentrations: 10^5, 10^6 and 10^7.

I serially diluted my stock all the way down to 10^-9. Under the hemocytometer, I counted an average of 30 cells at a 1:100 dilution factor. Hemocytometer is typical, at 10^4.

What concentrations will I have if i inject 10uL of 10^5/6/7 serial dilutions? What do I need to do to ensure I am injecting 10uL of 10^5/6/7 conc. into my organisms?

Feeling very dumb about this. 🤦

Please could I get some honest opinions on if my lack of data is insufficient to pass.

My project aim was:

create an armoured car T therapy for a specific disease: subcloning to create constructs, transducing T cells, testing expression then testing the effect of the activated T cell that also expresses this extra protein on the target cell line in vitro model to see any changes in the ‘disease’ markers.

My progress:
subcloning of constructs completed, sequenced and transduced into HEK cells to check basic expression.
Struggling with transduction of T cells, never made it to activation experiments and only an n=1 to show surface marker expression post transduction
Switched to Jurkat cells (immortalized cell line) successful transduction and proof of CAR- dependent activation.
In vitro models of ‘disease’ state did not work at an experimental level when adding Jurkats, no results here

Haven’t proven activation in T cells, no data of combining in a target in vitro model. Only 5 months left- in the case nothing else works how bad would this be data wise, would this be insufficient to pass? Supervisors much more busy and making no real comment on my sufficiency to pass, of course I will speak to them more on this.

Our academic lab is looking to switch our ELN from eLab to a less expensive alternative. A key requirement is the ability to maintain an inventory of biological specimens, antibodies, and plasmids (i.e. freezer/fridge/ln2 inventory), something which we currently rely on heavily in eLab.

LabArchives is a strong contender since we already have a university-wide license, but it doesn't include inventory management. We'd prefer not to use a Google Sheets/Excel-based method

Does anyone have recommendations for an ELN which includes an inventory or a stand-alone inventory that's reasonable priced (or free!) and handles lab inventory well?

I've noticed a - admittedly anectodal - tendency of wet lab biologists to be fat. Is this a common thing or just a coincidence? I'm mainly a bioinformatician and it is not uncommon for us to be fit. My personal theory is that sitting in front of the computer all day gives you a lot of pent up energy, whereas being in the lab all day is exhausting and makes you too tired to go to the gym.

Any thoughts?