Hey everyone! Has somebody of you wise lab rats ever put tube racks, like the standard colorful 5x16 ones, in the dish washer to clean? Some of ours are caked in grime (at least for my taste) and I am kind of not wanting to clean each and every hole by hand. Yes, soaking in water and/or ethanol makes it better already, but if I can avoid that… However, I am also worried they melt or bend under the heat of the dish washer. Please help a lazy lab rat! Have a great weekend!

Hi,

I work on synthesis. So I prepare silica slurry for column in an RBF. After loading the slurry, when I clean the RBF, the silica doesn't entirely go away.

So when I reuse the cleaned RBF for a different compound, I see small silica particles clinging to the walls of the RBF when I add any solvent.

I have tried a bunch of things-

-Wash thoroughly and repeatedly with soap and water and dry with acetone.

• blow air into the RBF after acetone as dried.
• keeping the RBF in the oven after cleaning and then blowing air in it.
• keeping it in the oven, and the washing the RBF after it cools for a bit and then rewashing it, acetone wash and then blowing air.

I still have the same problem with all the above steps I have tried 😭

Also, I don't see the silica particles when I am washing it with acetone. I see them when I reuse the RBF for something else while rotavaping it.

Any help is greatly appreciated.

I'm new to immunofluorescent staining but I've been staining mouse muscle cross sections for a while now with laminin. Recently, I have been noticing after mounting with DAPI (Vectashield Antifade Mounting Media with DAPI), it tends to be super vibrant on the perimeter of my section and less vibrant in the center. Is there any advice on how to correct this? Here is an example image. Thanks!

https://preview.redd.it/q0v15lnosb9d1.png?width=885&format=png&auto=webp&s=449799affd533e83530c4d8c5bf132ff21f26cc7

Hi labrats. I've been struggling for months with ELISAs that can detect analyte from conditioned medium samples, but not known analyte controls. Here, I'm using purified LALBA from human milk (>95% purity) in a human LALBA sandwich ELISA and the detected concentration is 0 - 18% of the known concentration.

I've ruled out the following potential explanations:

• model fit
• protocol deviation
• pipettor precision/accuracy
• platereader equipment/software functioning
• suitability of reference analyte (also works beautifully in westerns)
• handling of reagents; lack of freeze/thaw cycles

Additionally including a super outdated figure to help visualize the problem. I've now seen this problem replicated by 3 operators across 6 or so assay runs. We cannot be certain of our sample concentrations with a known analyte control. (We ran out of the milk control otherwise dilution of that would be suitable)

The ELISA also has this catch-all caveat: "due to the antigen specificity of the antibodies used in this assay, native or recombinant proteins from other manufacturers may not be detected by this kit"

As a final note, I don't think this is a popular ELISA and my colleagues and I did manage to recall multiple abcam and sigma ELISAs last year. I have so much frustration and uncertainty regarding assay accuracy and this is causing like a truth crisis for me.

If anyone has advice or has encountered similar problem, please help or assure. Thank you

Hey Lab Rats!

Have you ever been in a situation where you received two opposite recommendations on the research direction or experimental design you proposed, then you start to second guess yourself?

I’m proposing a new research project. Talked to my big boss (managing the whole research group) several times and big boss says “like the direction”. Then in a sub group meeting, my colleague (same level as me) says “strongly do not recommend going into this direction”.

Both me/boss and my colleague have valid reasons to support/not support with this direction, in the meantime the direction itself is something new and carries uncertainty/risks.

What would you recommend for handling this situation?

I’m struggling to find information on this. Can you direct me?

I got into resistance training in the last two years of my PhD (just defended the other week - wahoo!) I was mostly focused on bodybuilding, training about 4-5 days/week. However, I'm more interested in powerlifting these days and want to focus on strength and getting strong af.

I wanted to reach out to my fellow rats to see how they balance their powerlifting training and organize their schedule with lab work, data analysis and writing. In my experience so far, I feel that recovery is going to be an issue. It is likely because of the break from the gym I took while writing my dissertation, but my body is already wrecked from just one week of training ~65% 1RMs. I've been going in the evenings after lab work or writing for the day, but I'm so freaking tired by the end of the day, it's a real struggle and drag getting through my sets.

Anyway, if anyone has some thoughts, advice, or comments about balancing the their lifting and lab life, and recovery, I'd love to hear about it.

Hi all, hoping for some help with a PCR giving me trouble.

I’m trying to amplify different fragments from plasmids using PCR. Have 8 plasmids with the constructs shown in the graphic, with each plasmid around 3kb, but varied due to size of the inserts. Each coloured block is one of three different sequences that are assembled as shown and were ordered as synthetic constructs. The arrows represent primers (not to scale, 19-27 bp) and where they bind.

My problem is that I expected samples 3 and 4 to have multiple bands since the primers should bind to multiple spots, and I didn’t expect samples 7 and 8 to have multiple bands, as the reverse primer should only have one target.

Used Q5 DNA polymerase from NEB. Annealing temp was determined from NEB’s Tm calculator, and used 64°C for sample 1 and 67°C for the rest. Thermal profile used is shown. Used 200µM dNTPs, 0.5µM each primer, and 1 ng template plasmid DNA. Loaded 2µL PCR samples. Gel was 1.5%, ran 70V for 50 min and the gel was cast with HydraGreen.

Any thoughts about what is going wrong here, and what would you do to try and test these? Temperature gradients/primer concentrations etc?

https://preview.redd.it/f9yh4xk9ab9d1.png?width=1277&format=png&auto=webp&s=98ec777dcd6135e183feb4b0f85c5e8d6a2b9e67

Edited to add the image

What is your opinion about those cheap tips and tubes from Amazon? We buy most things from Fisher and Sigma but some of those consumables are just much cheaper and faster shipping from Amazon. But I sometimes wonder if we’re introducing some plasticizer like contaminants to our experiments with those tips and tubes of questionable origin.

Relatedly, do you get any lab supplies from Amazon?

Hello everyone.

I am currently troubleshooting my Western Blot and I am struggeling. I have a polyclonal antibody against CsrA from Thermofisher.

I get a lot of unspecific bands. Primary antibody overnight 4C 1:2000

The last three columns is the knockout mutant.

As everyone is aware of the power of ChatGPT, I found it useful for asking specific questions, summary a protocol in a paragraph or to check my text for mistakes but found I dont use it on a daily basis throughout my PhD.

Just yesterday a colligue was using another AI tool txyz.ai. This AI allows you to upload a pdf such as a reseach paper and it will give you a summary of the main results in the paper, more importantly it lets you ask specific questions about said paper. As I am currently writing my PhD thesis this is imensly useful to review papers quickly (btw I also validated that it gave the right conclusions from papers I have a good knowledge of).

So basically this lead me to the concusion of what other useful AI tools are members of Labrats using that I dont know exist? I though it be a useful thread for people to share AI tools they use and find benficial, as I am a firm believer in not gate keeping knowledge and if someone can make work esier for someone else they should as it benefits us all.

I have spent almost 3 years doing phd in Korea and I feel like I will no longer want to stay here. In these 3 years I started my phd 2 times in the different universities. What I faced first university was toxic PI. I used to work at least 9 to 10 without any weekend for one year and I ended that up. Plus that, that pi used to use research money illegally including student’s salary. (This is very common case in S.Korea and you know, university protects professors in this kinda things!!!) That time I was financially not possible to start over my phd in different country then I started my phd in different univ which is quite high ranked in the country.

Now, let me briefly explain my current phd situation here. Working hours and salary are not a problem. Instead, my pi is way too reluctant to meet his students esp. foreigners in the lab and we make meetings once in a month or two months (he says that he will do more meetings in the future). He is a ghost supervisor and almost not supportive at all. The most disappointing thing is he often breaks his promise of including me into a new project and, when that project comes to the starting point, somehow I am excluded. As I want to grow as a “real” scientist, I push myself searching and reading new papers and watch online lectures. But I feel like, I am just wasting my time without making not enough research results and products. I haven’t got my independent project so far and looking back, I have only contributed to other’s works. And addition to that, I already discussed with my current pi about my phd period which I want to finish within 4 years with 3+ papers and want to have my independent project, he was too arrogant to do that and as his thinking, he wants his students to do for 5+ years. With some achievements, of course I can do longer. However, as time goes on, one more semester has finished, I feel like that I have not developed anymore. All those years, what I see in Korea is like students are lazy, do not know what they are doing and professors are just running for money!!!

My original plan was to graduate PhD in Korea and then seek postdoc in the US. But I am quite exhausted now and considering looking for PhD in the US instead and regarding to that I have several concerns. Firstly, I worry that I might be too old (now I am 31) and I could graduate close to 40 if I start over again in the US (incl. app period, visa etc.). If I were in my 20s, I would absolutely leave and apply everywhere in the USA. Now I have a family, so I need to make a very careful decision. I wonder which way would be better and whether I should end my current PhD for the above reasons. My background is I have ielts 7 and first author of one small paper and co-authors of 3-5 papers.

So i'm beginning a position as a lab tech in a brand new lab and because its brand new while I am a lab tech on paper, I have a pretty significant administrative duty bordering on lab management (don't worry I'm being paid as such due to this) as I am the only one in the lab besides the PI. I think this is a unique experience to set this lab up for success (as there is no systems in place yet and PI is very open to suggestion) and was wondering what some veteran lab managers (or anyone whose seen a system that works) suggest for things like ordering and more specifically any organization systems for materials, etc.

I really want to do good at this job and impress my PI and also set up a system that will work for this lab and be easy to utilize in the future for others. Anything from how you would organize and keep records of samples like plasmids enzymes antibodies etc. to suggestions on proper ordering.

I have a lentiviral plasmid for expression of a pegRNA for prime editing. When the plasmid itself is transfected into cells alongside prime editor, it supports prime editing. However the lentivirus made from the plasmid does not. The obvious answer was an issue in lentivirus production or transduction, but I have assessed copy number by ddpcr (approximately 4 inserts/cell) and sequenced both the plasmid and the inserted lentivirus - both are correct. These conditions were done at the same time in the same cells. The only thing I can think of is an issue of quantity (but a single insert should be enough) or timing (lenti transduction was 18h before addition of prime editor plasmid).

We're all stumped - any thoughts?

What do these bright spots mean in the first 5 lanes? I thought it meant the gel was overloaded and degraded, however it seems that for the last 5 lanes the gel should be more overloaded since the rna concentration is higher there. In lane 6 till 10 I also saw bright spots at the place of the well, does anyone know what this is?

What's your opinion on people wearing shorts or skirts/dresses in the lab?

I work in a biomed lab and on our floor we've got all labs of the oncology department on our floor. It's (finally) becoming summer over here (Belgium) and I've noticed lots of people wear shorts/dresses and/or open shoes in labs and I am just completely blown away that people do this. One of the first things I was taught during my bachelors is to always wear closed shoes and long trousers for your own safety.

I guess I'm mainly interested in how it is in different places, do you gals/guys wear shorts or something similar while at work in the lab and what were you taught at school about this if anything?

Helloooooo everybody, I need someone who knows things about PCR and primers to weigh in. I have a list of primer sets from a paper that validated them for use against common viral targets in human samples. However, the paper did not include mention of how large the amplicons were for each primer set. I understand that under 200bp is generally suitable for qPCR, but you should try for more than 200bp if you want to do regular PCR and then run it on a gel. I am hoping to do regular PCR because my goal is qualitative. I need to demonstrate that my samples were not co-infected with other common viruses besides the target virus being studied. My question is how do you figure out the amplicon size of a target using the primer set? Especially when the target is a (-) ssRNA virus. Also, if the amplicon is less than 200, is it reasonable to increase the concentration of the DNA gel and run a regular PCR anyway? Thanks in advance. This is not my area of expertise and I'm on the struggle bus.

Hi, so I've been practicing submandibular bleeding on mice but I can't seem to get any blood. I poke the mice slightly above the dot on their cheek; no blood ever comes out. I would occasionally get the 1 drop of blood, but that's it. Any tips?

No diss to anyone here. You guys are all great. I have no hard biology or chemistry background. My MA is psychology. I have done some PCR and no western blots. I don't know what to do career wise....

https://preview.redd.it/sv65ml9nj79d1.jpg?width=514&format=pjpg&auto=webp&s=cf6a7d3a2cf7fec299554a63e5f8fce176514594

I am repeating a cell-based assay completed by an external lab (kind of method transfer).

Blue is my data.

Red is the data from the external lab.

We follow the same SOP.

Cells (and media) are the same but different lot#.

Drugs are the same and the same concentrations, but different lot#.

What should I modify the assay to have the same or close data to the data from the external lab?

Thank you!

I work in a very tight budget where at the moment we can’t afford to purchase all the necessary parts for Western (we have antibodies).

But we also want to publish, so that we can use that for grant writing (I know….)

If we want to “borrow” or “use” someone else’s “stuff” like all the washes in Western Blot, what is the most polite way to ask for it, and what is the most appropriate way to reciprocate for that?

🫡

I recently came across a person dissecting a mouse which was allegedly anesthetised. However each time a pin was used to pierce the limb to the dissection board, the mouse was visibly twitching and I could see the heart beating rapidly through the chest.

I have always dissected mice after euthanising them with CO2. Hence, I don't know if this is appropriate.

Am I being dramatic or is this normal after anesthesia?

Hey all, need some advice. I'm currently creating peptide libraries for expression in HEK293 cells. After ligation, I electroporate them into E. coli, plate enough colonies to ensure at least 100x coverage, then scrape off the colonies and maxiprep (I was told not to grow them in liquid culture, due to differing growth rates between members of the library). The peptide libraries are in a lentiviral construct, which then gets packaged and transduced into HEK293T cells.

With the throughput that I have, I can safely get around high 10^6 to low 10^7 transformants per library, which is good for libraries with about 10^4 members. Does anyone have experience electroporating larger libraries, like 10^5 or 10^6? Clearly, I could just do 10 electroporations (which would mean scraping about 200 plates), but I'm wondering if there's a better way.

I work in a BSL2/2+ lab with infectious agents, namely HIV and Herpes-B virus. I’m relatively new to this field and often feel like I’m contaminating my home with stuff from lab. When I get back from work, I always wipe down or spray my phone and backpack with Lysol or some other disinfectant. I also use specific shoes for lab.

These types of safety behaviors like spraying my bag and phone every day can get a little burdensome and I wondered if anyone else experienced something like this before or had some advice. I definitely am more on the cautious side in our lab and wear full PPE (gown, mask, goggles, gloves) whenever I’m doing any lab work. But I still do feel like my belongings get contaminated, primarily because I cannot account for the safety of my lab mates. I do sometimes see them bringing back contaminants to our desk space and while it may not be an every day occurrence, it does make me try to be extra safe.

Any advice or input is appreciated! Thanks so much!!

I did it for like 5 minutes it was like I was 5 years old again

I'm going to be sequencing an amplicon library (Illumina amplicon sequencing) generated by PCR'ing genomic DNA with an indexed primer pool. I was wondering what is suggested as far as amplicon size selection and clean-up. Would gel extraction with a Qiagen kit suffice? Does this interfere with the sequencing? Or would bead purification be prudent? I've seen that AMPure XP beads is often used for this, although you seem to be able to size select with the SPRIselect beads. I'd rather not spend the cash on beads if I can get away with gel purification. Any suggestions?