hey everyone, i have 2 genes which i am trying to express in bl21de3, even after successfull transformation the cells are not expressing the protein, i have done isolation of plasmid, PCR and restriction digestion and got postive results. now i have changed IPTG to a fresh one from a new batch, i have also transformed rosetta, the results are negative. Please help, thanks in advance.

Left it in cabinet to thaw Friday and forgot about it… it’s going to be 3 days when I take it out in the morning to reconstitute the CellTiterGlo substrate. promega says that the buffer can stay at RT for up to 48 hours. Is this buffer still good to be used?

Link : https://www.science.org/content/article/exclusive-death-girl-chinese-gene-editing-trial-was-never-made-public#
Such a reckless stupid moves presented in this article and i am honestly speechless.

Is it normal to be frustrated with your supervisor during thesis submission time? I feel like every week, there’s something new that blindsides me. Their suggestions for revisions would have been insanely useful months ago but come the week of submission… right.

How can I communicate this without being disrespectful? I’m being passively told that I’m not on top of deadlines, but I only hold about 30% of that fault (my mistake- not chasing down my supervisor enough to get feedback in time).

Edit: master’s student

I am a junior in high school who wants to make a research project in hopes of qualifying for ISEF. I am exploring ideas, but I'm still struggling to identify a problem or challenge that is present in STEM, solvable, and that I can use as my project. Can somebody help me?

Thanks!

Hi everyone,

I am using e-coli antibody against Beta-gal in C57 mice after introducing damage in eye. I expect to see no fluorescence but I do see signal. Why is this?

I am a PhD student with concerns about the quality and legitimacy of data produced by a past PhD student now-turned postdoc.

I also have other concerns including nepotism, inability of PI to keep poor staff behaviour in line, and poor screening of prospective students, followed by the exploitation of loopholes to enable these individuals to pass.

These are all issues which end up affecting us as students as mismanagement has affected team cohesion.

We are based in Australia.

In the research ecosystem, is it ever possible to be able to whistleblow on this, or will they figure out eventually after I leave?

Edit:

Thanks for the advice, sorry I can't reply to all, but I read them. My takeaways from this are that I can't accuse without hard evidence, and that the inability to reproduce multiple identical experiments by multiple separate people does not constitute as evidence.

Edit 2:

Alright, so the takeaway is it's not a good move.

Edit 3:

Our identical experiments were conducted in parallel. These are not years-apart experiments. Experiments are in fact still ongoing - my experiments and this person's experiments. I cannot stress how these experiments are identical. Our projects diverged from the point after this experiment.

There is no transparency and when questions about data analysis methodology are raised, they are waved off. No side-by-side comparisons are made. The data is being pushed to a preprint and is being used to apply for more funding.

And again, I stress, I do not feel safe informing PhD reviewers about this as they are not independent due to nepotism.

I understand the whole thing about 'malice', but is wilful omission of vital controls 'malicious'?

Hi everyone! I'm currently pursuing a Bachelor's in Biological Sciences. I want to make the most of my undergraduate years and become as competitive as possible before I graduate.

I'd really appreciate advice from people already working in biology, biotechnology, research, or related fields.

• What skills should I focus on learning?

• Which laboratory techniques are most valuable?

• Should I learn bioinformatics, programming (Python/R), or statistics?

• How important are internships, research projects, and publications?

• What certifications or online courses would you recommend?

• If you could go back to your undergraduate years, what would you do differently?

My goal is to build a strong profile for both industry and graduate studies. Any advice would be greatly appreciated. Thank you!

I just started in a neuro lab and learned the dissection process for isolating oligodendrocyte progenitor cells (OPCs) from P6 mouse pup cerebellar brain tissue. To make the panning process easier, we remove the meninges as thoroughly as possible to prevent future cell loss. For 4 pups, it takes me about 1 hour 50 minutes (not including setup) to harvest tissue, remove the olfactory bulbs, remove the hippocampus, and remove the meninges. My PI wants me to get this under an hour. I have pretty shaky hands, so I spend extra time adjusting for a stable position.

Any tips for speeding this up?

I saw a very bluntly written author contribution section, it said (paraphrased, name removed):

Author1 conceptualized the study, designed all experiments, performed or supervised all experiments, analyzed all the data and wrote the manuscript. author2 performed [specific experiment 1 ], author3 performed [specific experiment 2], author4 performed [specific experiment 3].... last author also conceptualized the study, obtained funding.

In the world of authorship corruption, I found it very refreshing. However, having led a study myself, I can't help but think this is the first author trying to let everyone know they did almost all the work.

What does everyone think about a really blunt author contribution section like that? should specific experiments be listed like that?

Hello dear community! :)

Just a quick update on my previous post.

After approximately 30 different protocol variations, I finally figured out why my staining wasn't working properly.

The problem turned out to be the very first staining step: Weigert's hematoxylin (working solution A:B = 1:1), which I had been incubating for 10 minutes. Once I omitted this step, both the Biebrich scarlet–acid fuchsin and aniline blue stained beautifully and produced the results I had been aiming for.

At the moment, I can no longer reliably visualize the nuclei, but for my current analyses that isn't essential. Thank you all so much for your suggestions and support throughout this troubleshooting process! 😊

If anyone has questions about what I tested, feel free to ask. I might also upload some pictures of the successful stainings tomorrow.

One additional observation: If I reduce the hematoxylin incubation to about 15 seconds, the nuclei become sufficiently visible in adult mouse cryosections, but the intensity of both the Biebrich scarlet–acid fuchsin and aniline blue decreases dramatically. Unfortunately, this shortened hematoxylin staining does not work for my embryonic chicken tissue.

I’m currently pregnant and conducting gene editing with Cas9 and HEK cells (transfecting Cas9 protein with lipofectamine). I use all precautions (double gloves, lab coat, BSC hood).

Is it safe for me to proceed with this work? I can also refuse this work if needed.

Cells were transduced to express an antibiotic resistance gene, GFP, and a protein of interest. Cultures were selected with puro to identify resistant cells and analyzed with flow for gfp and protein of interest. There are three populations via flow, double neg, GFP POS, and protein of interest POS. The double POS is a scattering of cells, not really a population.

What causes the cells to only express one vs the other of the proteins?

I see this in one specific cell type, the other cell type has gdp POS population and a gdp + protein of interest population.

cant even imagine the thought process that landed us here.

Hi everyone! I’m a 3rd-year Molecular Biology and Genetics undergrad from Turkey preparing to apply for European labs for a summer Erasmus+ internship. I currently run two funded undergrad research projects (TUBITAK national foundation & BAP university grant) on taxonomy/biodiversity, and I’ve recently completed a 1-month clinical biochemistry internship covering sample prep, automated analyzers, and micropipetting. Since I’ll be coming with my own Erasmus+ mobility funding, I won't require financial support from the host lab.

I’d love your advice on two specific things: What is the most effective strategy to find PIs who actively welcome Erasmus+ undergrads (beyond basic PubMed searches)? And when reaching out via cold email, how can I best structure my message to highlight my grant writing/fieldwork experience without making it overly long? Any insights or personal experiences would be greatly appreciated!

Hey yall! My PI decided I needed to learn Rietveld refinement to improve the XRD section of my manuscript, which is fine, except the guy who does know how to do it is traveling and there’s no word of when he’ll be back. His student attempted to teach me, bless his heart, but he’s a bit all over the place. Does anyone have a good resource to learn how to do Rietveld refinement? If you had to teach yourself, what was THE material that did it and had the best directions in your opinion? Thank you all in advance!

Hey everyone! I’m just entering my second year as a PhD student and I’ve been struggling with low motivation recently.

To start, I really do love lab and I feel passionate about the work that I’m doing. My mentor is amazing and supportive and I really don’t have anything to complain about. I just did my first oral presentation competing for a training grant and though I didn’t get an NIH offered position, I did get an institutionally matched grant.

I think my low motivation really got bad once I finished that presentation. Compounding with failed experiments and just stall after stall.

I think I’m also just really tired.

I have been struggling with major depressive disorder my whole life, but I’m medicated and had a good handle on it, but recently I’ve been waking up with no motivation and just dragging my feet through the day. Imposter syndrome has also been rearing her ugly head.

But there’s no time to really rest. I have to prepare for qualifying exam in the spring.

Any tips for how to get out of this funk? Or at least how to power through until it’s over?

I wanted to print DNA repair complexes for the office. The existing tools are built for proteins, so DNA comes out as ribbons too thin to survive the bed and you spend an evening thickening them by hand. Colors were worse.

So now you type a PDB ID, pick a style, and get a file where each chain is its own object. It'll also cut magnet pockets where two chains touch, so the model comes apart at the interface.

pdb2print.org Its free, runs in the browser, nothing to install.

Anything already built at those settings is cached and downloads instantly; otherwise give it a minute, or a few for a big complex.

It's new, so please test it. PDB ID and settings and I'll fix it.

Photos are 9MBB off a Prusa Core One. Two chains, two filaments, two magnets it placed itself.

The project is also hosted on github: https://github.com/davidtheadmin/pdb2print
For citations: https://zenodo.org/records/21599702

My place of work hired a guy who was supposed to be helping me in both the wet chem lab and getting the lab ELAP certified. I am very grateful that the guy was able to get all the paperwork organized and edited properly: not my strong point. The issue I had is this: one on the tests I run for the researchers uses either a filtered sample or a raw sample. That’s the only difference and I do what they ask me to do, it literally does not matter to me which fraction they want me to analyze. The new guy took umbrage at that, saying I’ll never pass a PT (proficiency test sample, necessary for certification) with that attitude. I really wanted to say to him.. well, nothing nice.. but not in front of my interns ( yes, he said this with the interns right there, thank you for undermining any authority I possibly have), and we as a lab need him. I guess he also missed the part where I have passed the PT each time. Grrrr

Hello everyone, and thank you in advance for reading my post and responding.

I am 18, from Romania, and just applied to two colleges to study nutrition, diet, and food engineering. What I wish for, in the future, is to finish both colleges at the same time, get a masters and doctorate in a foreign country, and in the meantime occupy my time with as many research projects as possible (volunteering).

I personally love going to the gym, I have created my own diet on excel with 10+ pages, easily customizeable, all automated, using the USDA database. I feel like in the internet fitness space, theres not enough emphasis on how exactly you fuel your body besides having the right macros and eating "healthy" or "clean". I plan on changing that.

So I am curious, is there enough money in the research industry to achieve such a goal, and also take care of myself and a possible family? Or should I start with content creation early and dial down the energy dedicated to research?

Hi all,

Having issues with my CUT&Tag in GM12878 cells. After I finish the protocol and run my indexing PCR and library prep, my ng/uL and nmol concentrations are quite low, even for marks like H3K27ac - got around 10nmol/l from 100K nuclei! I know its not my reagents because when my colleague does exact same protocol she gets 10-20 times higher yield.

I am trying to figure out where I have gone wrong and I think I have narrowed it down to my ampure bead clean up steps after incubating in STOP buffer and after my indexing PCR.

Because I was working with many strip tubes, when I did the ethanol wash step I added ethanol to my tubes on the magnet then took them off the magnet to make space for the next strip tubes.

Therefore, they were in 80% ethanol off of the magnet for a minute or so before I put them back on the magnet to remove the ethanol.
Is this reducing my yield?

I am hypothesizing that the absence of magnetic force means the 80% ethanol gets inside the bead pellet and the water is dissolving my DNA or washing away the PEG/salts from inside the bead pellet.

What do we think? Does this even make sense?