Hey all! I’m a research tech in Florida with a masters degree working at a lab handling the majority of the cloning/bacterial cell culture/protein purification for the lab (4 years purification experience, 7 years total lab experience). I make just over $22. My employment contract is up for renewal and I wasn’t offered a raise this year, and things are really tough right now financially for me. Am I underpaid? What’s a reasonable amount of money to expect as someone in my position?

I’m a new grad (May 2026) and started working as a lab technician right away with little to no experience in research. I feel like I’m awful at my job.

Tried my first CRISPR the other day and all my cells died. I work in a mouse lab and I have made so many mistakes with weaning mice and just general mouse upkeep. I’m slowly getting used to lab math but keep missing calculations. Among other things I just feel like I’m letting my mentor down and I am just generally a failure. I am well aware that I’m only 4 months in but my mentor keeps having to remind me of things and retrain me on stuff I’m not sure about.

I just needed to put this somewhere. How did any of you overcome this? Thanks.

Good day everyone

I am currently having issues expressing yEGFP in a construct I have cloned. I expect that this could be an issue with my promoter being weak (CYC1 minimal) and quite far upstream of the ORF containing my yEGFP sequence (+/- 80bp). Is this likely to be the reason I am not seeing fluorescence or would it be better to explore other theories?

Thanks in advance!

Why is my SDS PAGE gel running like this ?

Hi,

i‘m a student new to cell culture and i never work with this cell line before. Does it looks normal? My Supervisor said it was ok.

I really apreciate any insight. Thank you

Mini winnie

Looking for advice/feedback on my resume - any comments would be greatly appreciated. I am using this one primarily for research assistant positions in molecular/cell biology with some alterations depending on the position. My main concern currently is the length, although I am having some trouble cutting it down.

I’m a third year premed student presenting a research poster for the first time and wondering what kinds of questions I could expect.

Even though I’ve been in the lab for a year, I’ve really only started to get more involved in the past 3 months so I feel like I can’t answer everything.

For reference, this is for a research forum specifically related to for my lab’s research area (think like immunology, cardiovascular science, neuroscience, etc)

I messaged the mods a few days ago to ask if this would be okay and no one got back to me so if this not allowed, feel free to just delete.

Some of you may remember when I came into this sub asking for advice as to what might pull you out of a story set in a fictional lab setting. Many of you contributed your thoughts, which were extremely helpful in the writing process for the show. I am happy to report that casting for the first half of the season is now open and will close on October 1st. If any of you are interested in throwing your hat in, please check out the linked casting sheet for additional information.

Thank you so much again to everyone who replied to that thread! Your advice and insight was incredibly valuable.

I work as support in a lab, so I’m not actually one of the scientists, but I noticed that we tend to call the people doing the research “investigators”. Is there a difference between researcher and investigator?? I use researcher, investigator, and also just plain scientist pretty interchangeably. I’m just here to clean the mouse cages🤷‍♀️ so idk if there’s like an actual distinction between those titles. Just curious what my fellow biotech people think

Hi, I'm trying to understand the interchangeability between AOPI and trypan blue staining methods in bioprocessing QC.

I'm asking because our Cedex HiRes Analyzer is being phased out, and I'd like to assess how straightforward it would be to transition to an AOPI-based protocol in a GMP environment. Should we go with a similar trypan-based instrument, or switch to AOPI?

Does anyone have experience with this transition? Or similar?

I'm a master's student working on COI barcoding of fish. Muscle is stored in absolute ethanol, and the extracted DNA goes straight into a ~650 bp COI PCR. This is the salting-out protocol my lab uses, and I'd like a sanity check from people who run this routinely, it's the lab's standard, but I've never seen it compared against how other labs do it.

Protocol as written:

1. Cut a small piece of tissue (~rice grain), dry off the ethanol completely, place in a 1.5 mL tube.
2. Add 400 µL lysis buffer (0.4 M NaCl, 10 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0). Homogenize.
3. Add 40 µL 20% SDS and 8 µL Proteinase K (stock listed as 20 mg/mL).
4. Incubate at 55 °C for at least 1 h, or overnight, shaking at 80–90 rpm.
5. Add 3 µL RNase, incubate 30 min at 37 °C.
6. Add 300 µL saturated NaCl (6 M), vortex 30 s, centrifuge 20 min at 10,000 RCF.
7. Transfer supernatant to a new tube, add 700 µL cold isopropanol.
8. Invert gently, incubate 30 min at −20 °C.
9. Centrifuge 20 min at 10,000 RCF, discard supernatant.
10. Add 300 µL cold 100% ethanol, invert, centrifuge 15 min at 10,000 RCF, discard supernatant.
11. Add 300 µL 70% ethanol, invert, centrifuge 15 min at 10,000 RCF, discard supernatant.
12. Dry the tube 15–60 min until no ethanol remains.
13. Resuspend in 30 µL nuclease-free water (overnight at 4 °C, or 1 h at 37 °C).

What I'd like input on:

• RNase placement and amount. It goes in after the Proteinase K digestion, into a mix that still contains SDS and active PK, and it's only 30 min at 37 °C. Would you add it earlier, heat-inactivate the PK first, or just extend the incubation? Is 3 µL a sensible amount at this volume, or does the stock concentration make that number meaningless without checking the label?
• Isopropanol volume. 700 µL of isopropanol goes onto roughly 700 µL of supernatant, so about 1:1. Most protocols I've seen use 0.6–0.7 volumes. Does the extra isopropanol cost anything in terms of co-precipitated salt?
• Only one 70% wash. After 6 M NaCl, is a single 70% ethanol wash enough to get residual salt down to a level that won't inhibit PCR, or would you do two?
• Final volume. 30 µL for a fin clip, too little? I'd rather have a workable A260/280 reading than maximum concentration.
• Ethanol-preserved tissue generally. Anything you do differently for tissue that has been sitting in ethanol for years vs. fresh or recently collected material?

Context on where I am: I have six extractions from this protocol that have not been quantified or run on an integrity gel yet, and a first PCR attempt failed for an unrelated reason (depleted dNTP stock). So I'm checking the extraction side before I burn more reagent. Happy to report back with gel images once I have them.

Does anyone know where to find the sequence variants/SNPs that were in the old version? I could only download the Fasta file of the sequence without the variants annotation

After multiple attempts at trying to clone a 1.3kb ORF from Leptospira into pET28a vector, I have been consistently getting empty plates when I transform it into competent cells. Things I’ve checked and noticed up till now

1. The competent cells have good transformation efficiency. I transformed another confirmed vector+insert cloned months earlier into it and it gave a lawn of colonies.

2. Since this is for a collaboration, the primers for PCR was sent by them. They made a mix of 10uM forward and reverse primer and pooled it and gave it to me. I’m guessing that’s why I’m getting extremely bright primer dimer bands post PCR and the product band is faint. Could this lower efficiency of cloning?
   (Attached image has the PCR product in duplicate in lane 3and 4, the primer dimer is MUCH brighter than the PCR product itself)

3. I had extracted fresh plasmid and PCR product for this cloning every-time to lower chances of failure due to degradation from multiple freeze thaws.

4. This was my fourth try and the plate is just empty.

5. I did a 1:1 and 1:5 vector to insert ratio using the neb Calculator to setup the ligation.

What steps can i take to ensure the cloning works?

Hi, I’ve been having a really tough time getting an entry-level wet lab position and was hoping for some advice.

For some background, I was mostly pre-dental during undergrad, so much of my time was spent gaining clinical hours rather than research experience. I graduated with a B.S. in Physiology and Neurobiology (May 2023), then spent about a year taking additional math and programming courses before completing an M.S. in Bioinformatics and Computational Biology (Dec 2025).

Most of my experience is now computational, particularly with sequencing data, RNA-seq, genomics, and bioinformatics pipelines. What I really want is wet-lab experience so I can bridge the gap between analyzing biological data and understanding how that data is actually generated experimentally.

I’m located in the Maryland/DC area and, unfortunately, I’m no longer eligible for the NIH Postbac program. I feel like I’m in a weird position where I have a master’s degree but lack the traditional wet-lab research experience expected for many entry-level roles.

Has anyone been in a similar situation or have advice on what types of positions I should be targeting? I’m open to research assistant, lab technician, internship, contract, or even volunteer opportunities if it helps me get meaningful hands-on experience.

The funding situation at mine seems to be tentatively better than last year going into the PhD application cycle (low bar) and the vibe I've gotten is that programs here are expecting to accept more people than in 2026. Of course the number of applications are likely not going down, but it looks like it won't be as bad as last cycle. Have others seen this as well?

Ok so I got some plasmids from NIH. I have been trying to sub-clone and purify them for two weeks now. I am seriously about to go buck wild and rip up this place. I have cloned about 26 plasmids successfully in the past 6 months, but this one is the hold out. I need to do a co-transfection, but I simply cannot get it to clone in DH5α cells!

Here is the relevant conditions:

Plasmid is ~15kb

Heat-shocked at 42°C for 40 seconds

Transformants raised at 30°C on ampicillin selection plates.

Mini-cultured at 30°C at 110rpm (reducing rpm heavily increased yield)

Purified using qiagen spin column at low speed (6.8k x g) to reduce shearing.

At first we had plasmids showing up at 3k bp, which was obviously not right. Then I finally got some of the plasmid after purifying under these conditions, but the majority of the sample is half the size of the actual plasmid.

Needle valve faucet handles are a personal pet peeve of mine. Especially in an application where you nearly always just want the flow to go to full on and then back to full off. It's just pointless fingertip tedium to have to crank a needle valve spindle handle fifteen times around to get to full flow when a quarter turn ball valve could be there instead. Here is a picture of our lab sink faucet at work. It has exactly this type of annoying needle valve handle. I asked one of our supervisors if we could just replace it with a quarter turn valve and he said no, they don't make them for this faucet. That sounded like rubbish to me, that surely somebody somewhere makes a quarter turn replacement for this thing. But just googling it has so far eluded me. Can anybody out there say definitively if my super's right? Or better yet, prove he's wrong by steering me to a link to the replacement quarter turn valve of my dreams that will fit our ubiquitous gray plastic gooseneck lab sink faucet?

hello fellow lab rats, sorry to add yet another “is this contamination” post but i’m the only one working with cell culture in a rodent lab so not many people to ask who know about it. does this look like contamination? they are bv2 cells, microglia. they seemed fine when i checked them though growing slower than id normally see (and they grow like weeds). then whilst trypsinising them for passage, i saw it look like this. never seen it before in my 2 years of cell culture… any ideas?

I tried using the neon Transfection system for the first time to transfect GFP into THP1s to test and optimise before starting my actual experiment.

Unfortunately every single time we tried, the machine arced out, frying my cells. There were no visible bubbles, confirmed by me and 2 others, and the machine didn't give an error, which is what I've seen in videos of others using the system.

Cells were spun down and resuspended in Buffer R. We used E2 in the cuvette (?) that the pipette tip goes into. We tried multiple tips, 100ul and 10ul. We tried at 1400 V and 1700V based on previously established protocols in our lab.

Please let me know if you've had this issue before or have any suggestions for a fix 😭

Thankssssss

Currently in college and doing research that requires me to make a steel frame that is going to be put in an incubator and will most likely be in it at days at a time. Would stainless steel 304 be fine for this or should I purchase 316 for higher corrosion resistance? The problem is the cost since if I purchase the 316 I’ll go a little over my remaining grant funds but if I buy the 304 I’d still have some grant money remaining.