Hello, I have been doing bradford assay and i keep on getting negative conc. What might be the reason for that? I use lab-made bradford and BSA. Honestly, it is also my first time so I don’t know based on what i should determine whether the data i am getting is accurate/ realistic or off, so I would appreciate the advice.

Im using a nanopore sequencing service that works very well 90% of the time. 10% of the time it helps to be able to look at the data myself and also perform some de novo assembly.

Does anyone have suggestions on which assemblers to consider?

I started with Flye, since it can run in Geneious and I am running into issues. It often won’t assemble, and I’m not sure if it’s because Flye was developed for much larger contigs/reads.

Full disclosure, I work at a research data management software company, but I’m not trying to sell people on it.

What are people willing to pay for software that helps them manage their sample storage and assay data? I’ve talked to private and publicly-funded labs, and the price sensitivity seems to be largely determined by whether they are private or public.

Hi guys, so I’m not here to rant. Actually looking for a general opinion.

I just worked as a lab tech in an academic lab for two years before getting a job at a new lab in the same university. I’m taking 1 grad course at a time and preparing for a PhD application this fall. At my last lab, my PI had me do a set of western blots. The westerns were for 6 proteins in 3 different samples. I did all the lysis work, BCA, and western from start to finish. I did some experimenting with different brands of antibodies for these proteins, and experimented with different incubation times to try to get nice clean bands. I did this western like 7 times. My PI took the data from the best 3 images. The PI mentioned (n=3) on the paper. I ended up leaving the lab to take a higher paying tech job after my two year mark. A couple months into this new job, I see a “pre-print” publication from my last lab with my westerns on it, but I am not mentioned anywhere. I’d really like to be mentioned cause I don’t have any publications and I’m applying this fall for PhDs.

Should I reach out and ask if she’s willing to put me on the publication? Is it normal to have your data published and not be mentioned? Thoughts? Tbh I’m upset because I thought I’d be a co-author or something.

Thanks!

I told myself that whether or not I got it, this was going to be the last neuron for the day. Came through under pressure, thank you gods of patch clamping!

Title. The TAQ PCR file got deleted and I don’t know what settings for a protocol to run

Hello,

I am a part of a multi-omics project involving single nuclei RNA, ATAC, and ChIP-seq. I am completing the RNA portion, but have been tasked with isolating nuclei for all three analyses. We're working with frozen cattle skin biopsies (~50-100mg each) which is a relatively tough tissue to homogenize and lyse effectively. I first tried using the 10x kit, though the lysing reagent didn't work well with the skin tissue we have. After some failed troubleshooting attempts, it was decided this kit was not compatible with our sample type. I am now trying to create my own protocol for this. Here are two lysing recipes I recently tried with some results (although not great):

-1M HEPES (pH 7.5), 1M KCl, 1M MgCl2, 10% Tween-20, 10% NP-40, 1M Sucrose, 10mM EDTA, 1M DTT, nuclease free water

-1M Tris-HCl (pH 7.4), 5M NaCl, 1M MgCl2, 10% Tween-20, 10% NP-40, 5% Digitonin, 1M DTT, nuclease free water

Nuclei were stained and measured with AOPI on a Celldrop cell counter. I think I need to adjust the settings for counting, but I wanted to know thoughts/comments on some of the images. Are these actually nuclei I am seeing here? It seems there is a lot of debris, but previously I was getting no red whatsoever, so I'm hopeful this time I did actually get something.

If these are nuclei and not false readings, I need to optimize to increase the count. Currently I am using plastic tube/pestles for homogenization, but I am thinking a glass dounce homogenizer might work better for this. I doubled the amount of lysing reagent and extended the incubation time slightly. Some have suggested using collagenase, but I read this is most effective at 35-37C. Since I am completing the RNA-seq portion, I have been doing all reactions on ice/cooling tubes on dry ice, so I'm not sure how I would include this.

Any advice or suggestions would be greatly appreciated!

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Hi. I graduated with a BA in Poli Sci, and really want to get into a master's program for Psychology, and hopefully get into a phd program for clinical psychology. I have only 11 months as an undergrad RA in a social developmental psychology lab. I have applied to a lot of research assistant positions related to psychology, but can't find a single job. I got one interview and then got ghosted. Any direction/help would be appreciated.

I went to ASM last year and some of the people in my program had an insane amount of merch. I’m talking, they went to drop off bags at their hotel and came back for more. But I feel like while I was going around, the people were being really stingy. Is there a secret to scoring merch??

I am a member of the eyewash club back when I accidentally splashed dirty bleach water in my eyes at my old lab while cleaning dishes.

Since I am going for my Master's, I wanted to treat myself to some splash goggles that are prescription.

I saw Stoggles, but I do not think they are splash safe, more like Z87.1 kinda goggles. What do you guys recommend?

(I’m talking about the main knob on the top of the tank not the dial on the regulator that you turn to start CO2 flow). Is this normal? Do I just let it release and see if it stops? Is this normal? I’m obviously a little scared to do that……

Basically it wasn’t doing that but then I took the regulator off (to get rid of the tank bc our lab is decommissioning) & then put it back on (bc I realized I had some more mice to sac).

I been in a lab with this item in the past, i want to recommend it to my current lab, but they never heard of such a thing and I can't find the name of it. I want to call it a micro-injector, but according to Google, that refers to something else entirely. It's the size of a micropipette; you draw up a relatively larger volume; my former lab used it for DNA loading dye. Then you press a button, which shoots out a set amount of the liquid. it is very efficient when you need to pipette loading dye into a lot of samples.

What exactly does tenure ensure for PIs? What sort of stability and changes occurs from assistant to full professor?

Like I know its powerful and all that. But holy shit it might be the most unintuitive bit of important software that anyone needs to use anywhere. Like fuck me, I just want to grab exons with like a 100bp pad of introns on either side. I feel like the end of 1984 or Clockwork Orange staring at the screen.

We have everything she would need, I just don't want her to be on the switch all day.....

Let me start by saying that my knowledge of bioinformatics and statistics is not great. I’m designing a site saturation mutagenesis screen where I am going to eventually mutate 3 codons in my protein-coding gene of interest, clone into an expression plasmid, maxiprep, then use the mutant plasmids to produce adeno-associated virus to transduce mammalian cells. I will use the mutant protein-expressing mammalian cells for my actual experiments.

Because there are 3 codons being mutated, there are 32,768 ( 32^3 ) equiprobable variants, so based on GLUE-IT calculations, I need a bacterial library of at least 468,500 colonies to be 98% sure that my bacterial library is 100% complete (the minimum my PI wants). I understand this.

What I am not sure about is how many mammalian cells I need to successfully transduce from this bacterial library to now be 98% sure that the transduced mammalian cell library is 100% complete. Do I essentially now have 468,600 equiprobable variants and need to transduce a minimum of 7,947,000 cells? I honestly do not know how to devise this and would really appreciate the feedback of you great people.

Literally told me he can’t and won’t, he wants me to be independent fully. I just started my PhD. Is this a norm? No postdocs in the lab. Should I start looking for other labs?

Hi guys I did something dumb. Working with pBAD for the first time and im also expressing in bacteria for the first time as well so I was super excited! I asked a post-doc about how to prepare and he emphasized using BL21 for my expression so I did...until i did a quick google search oops. I already transformed into BL21 without thinking.

Our lab doesn't work with TOP10 or LMG194 so we need to order it for comp cells, but is protein expression still possible? I understand that TOP10 and LMG have deletions where they do not metabolize arabinose, but I might just try to express for the fun of it until we get TOP10 on our hands.

I’m using a fluorescent microscope for my PhD, and I have hundreds to possibly thousands of cover slides to image. This means countless hours of work in the dark. I get motion sickness very easily, I last about 10 minutes if I play video games or if I watch a movie in the car. About 50% of the time, when I take images using the microscope (and I have to look down it to focus and choose which cells to image), I will feel nauseous almost instantly and the feeling lasts all day. If I try to power through, every single time I move the slides and am staring down the eyepiece until I see a clump of cells, I feel this awful pang of nausea and headache, which gets worse the longer I stay on it. The longest I’ve managed to work today is 40 minutes.

I haven’t talked to my PI about it, but even if I do, I doubt there’s anything he can do about it… it’s my PhD and it’s too late to change topics now, with just over a year left. Nobody will be taking them for me, rightfully so as it’s nobody else’s job. What should I do? Does anyone have a similar issue?

Hey guys :)

I work for a biotech company and we have this BSC that’s used once a month for grow promotion testing of our petri plates (Quality Control). The BSC is run continuously and I’m asked to define an appropriate cleaning frequency. Is cleaning only before and after use enough? Using bleach wipes followed with IPA With cleaning under the surface of the BSC quarterly

Are there any guidelines, regulations we should respect? Thank you

Hello.

Like the title says, our lab's fluorometer is getting a reading of the DNA standard in our Quantifluor ONE dsDNA system, but the standard is curved instead of linear. Does anyone have experience with Quantifluor and know how to fix this?

I thought about adjusting the dilution because the recommended dilution starts with 1 part DNA, second dilution is 1 part DNA and 1 part TE, and the remaining dilutions are 1 part DNA to 3 parts TE. It seems a little wonky to not have a consistent dilution pattern, but what do I know. Any helps towards this would be amazing!

I'm an ex-labrat who's trying to make a tool that makes your lab life easier but I think it's better to ask the people what they would find more helpful (as I need to start with something and I want that something to be useful). so, here goes: what would have a greater impact on your lab life?

1. Querying your research projects and external databases with AI tools

(think of connecting chatGPT-like thing to your lab notes and data - all supersafe, no hallucinations, but like ctrl+F on steroids)

2. Automating note digitization with a predefined structure.

(think of voice notes, taking pics of handwritten notes etc as input and having a nice, properly formatted lab notes in an electronic lab journal of your choice)

edit: grammar

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Hello fellow lab rats,

I’ve been wondering if anyone here could share some useful tips with me for conducting ELISAs from scratch (aka using homemade antibodies, homemade prepared standards, etc.). I’ve made quite a progress with the assay but what bothers me is that even though I always conduct the assay in the exactly the same way, there are discrepancies in between the standard curves I make. It’s not a big difference, sometimes only with absolute values, sometimes the slope of the curve is slightly different. Is this normal when I’m preparing everything from scratch or could there be some technical mistake that makes the curve fluctuate this way? I’m using a plate washer for washing, so this should not be a problem.

Just to add, I always dilute my standards in a way that I transfer at least 20 uL never less and I have diluted stock solution from which I always dilute the standard curve solutions. Could these discrepancies be caused by dilutions of detection antibodies? The used dilutions are rather high 1:5000 and 1:10000 so I always transfer 0,5 or 1 uL of antibody stock solution (which I know is not precise but since these antibodies are used in excess anyway, I figured it shouldn’t be such problem). Do you have any tips how do you dilute your antibodies to such high dilutions?

Thanks you for reading and sharing any tips you might have!

Hi! So, maybe I fucked up by putting FBS inside a fridge for a week or two, when I’m supposed to put it in a -20 C freezer? Would it still work fine or not? I’ve been using an expired bottle of FBS before this bottle because that’s what our lab had and it worked fine so… I don’t know actually.

Edit: for context i’m working on my undergraduate research