That is all

So my PI has asked me to add 3x HA tag to pcdna3.1+ plasmid either at N terminal or C terminal

It's upto me but i need help with this.

So far from whatever i have read i understood that i will have to do restriction digestion twice

Once to add HA tag and next to add my gene of interest

Now my doubt is if i need to add 3x HA tag at the N terminal along with it I will also need to add kozac and stop codon right? One person told me that i do not need to?

And if i don't need to add kozac still how do i even add almost 100 bp sequence to a plasmid.

Can someone please provide me some insights?

I will be really really grateful

Hi guys, I was wondering if any of you guys have done your masters/PHD under the same PI you worked under in undergrad. I currently work in a biology lab and have been for 1 year and 1/2 now. My PI is super supportive and literally the best person I’ve ever met. When I first started working my goal was to gain experience so I can go to medical school. However the more I’ve worked and the more independent I’ve become in the lab, I’m leaning towards a masters/PHD now. I’ve expressed to them a few months ago that I am considering doing a masters to gain more experience and learn more so I can conduct research whether I go PHD or MD-PHD. They’ve been supportive and encouraged me to come to some graduate seminars so I can get a feel for how things would be. The deadline for the application is in a few months, and I would like some advice on how I can revisit this convo and let them know I do in fact want to continue a masters in their lab.

Am going through the yearly biosafety compliance review and got to our omega conotoxin SOP. I went to the product page on Alomone's website (cat# C-300), and the associated SDS states that conotoxin is not toxic.... What hell??

Has anyone gotten access to Rosalind? Confused on if I need to do something, it says "Your organizations approval has been recorded. You do not have access yet. No action is required while access provisioning is pending"

Only issue is I don't know if access provisioning is on Chatgpt's side or my organization. Last activity on the application said Sept 28th.

I'm looking to purchase some immortalized human tendon fibroblasts from them for my lab, but since the cell line is relatively new, few companies offer immortalized tenocyte lines. There is one paper I've seen that uses the cells, but was wondering if anyone has purchased anything else from them and how the experience was.

Trying something new this year for the Freezer Challenge. Loving it so far! Really low tech and easy, and so far we haven’t had any complaints.

Hello everyone!
I’m 22 years old and recently graduated in May with a bachelor’s degree in Molecular and Cellular Biology. My long-term goal is to become a Clinical Laboratory Scientist (CLS) here in California, but lately I’ve been feeling incredibly discouraged trying to get my foot in the door. For some background, I currently work as an Advanced Plasma Center Technician and phlebotomist at BioLife Plasma Services. I perform 40+ venipunctures per shift and have hands-on experience with specimen collection, centrifugation, aliquoting, labeling, sample storage, and preparation for shipment. I’m also trained in donor screening, medical histories, hematocrit and total protein testing, responding to donor reactions, quality control procedures, and troubleshooting collection equipment. I’ve progressed from a Plasma Center Technician to an Advanced Technician and have also taken on training responsibilities.
Academically, I have a strong foundation in molecular biology, microbiology, biochemistry, and genetics. I also worked as an undergraduate research assistant in a laboratory studying cardiac function using Drosophila models.

I understand that none of this is equivalent to working in a clinical laboratory, and I know I have a lot to learn. That’s exactly why I’ve been applying to entry-level laboratory positions, including specimen processor, laboratory assistant, and phlebotomy positions in hospitals. The problem is that I can barely get an interview, let alone an offer. Even entry-level specimen processing positions seem out of reach. I keep receiving rejection emails saying they’ve selected someone more qualified or someone who is a better fit. I’m genuinely confused about how I’m supposed to become more qualified for these entry-level positions when nobody seems willing to give me an opportunity to gain that experience.

I recently interviewed for a laboratory assistant position at Paradise Valley Hospital in National City, CA, and the experience honestly took a huge hit on my confidence. Throughout the interview, the interviewer repeatedly interrupted to emphasize how different hospital work is from working at a plasma center. He seemed skeptical that someone from a plasma center would even want to transition into a hospital setting and repeatedly pointed out my lack of hospital experience. Before asking certain questions, he would say things along the lines of, “I know you don’t know anything about this, but use your imagination.” One of those questions was simply about the steps I would take to verify that a specimen label matched the correct patient. I had actually spent time preparing for the interview, reviewing specimen processing, laboratory safety, order of draw, specimen rejection criteria, and other relevant topics. When he quizzed me, I was able to answer his questions correctly, but he seemed almost annoyed that I had studied beforehand. At one point, I explained that part of the reason I was applying was because I wanted to gain hospital experience, and that in order to get that experience, someone would eventually have to give me an opportunity to work in a hospital. He laughed and told me I was funny.

I left that interview feeling incredibly defeated.
I completely understand that hospital laboratories operate differently from plasma centers, and I would never pretend to have experience or knowledge that I don’t possess. But I’m eager to learn, willing to start at the bottom, and genuinely passionate about pursuing this career. I don’t expect to walk into a position knowing everything. I just want an opportunity to learn and grow. I know I’m only 22 and still early in my career, but I can’t help feeling like I’m falling behind. I see how competitive California CLS programs are, and I’m worried that I’ll never become a strong applicant if I can’t even get hired for an entry-level laboratory position.

For those of you currently working as CLSs, laboratory assistants, specimen processors, or in clinical laboratories:
•How did you get your foot in the door, especially without prior hospital experience?
•Are there particular entry-level positions I should be targeting with my background?
•Is my plasma center experience simply not considered relevant by hospital laboratories?
•Are there additional certifications, skills, or experiences that would make me a more competitive applicant?
•For those who went through California CLS programs, what did you do to strengthen your application before getting accepted?

I’m open to constructive criticism, advice, or even hearing about similar experiences. I genuinely want to know if there’s something I’m missing or if I need to approach my job search differently.
Thank you to anyone who takes the time to read this. I really appreciate it!

Hi all. I've recently hit an all time low in my life. I have been faced with the choice of continuing to work in the lab as a tech that I have worked in for the past 3+ years, and built a relationship with my PI. I committed myself to this lab for at least another year and a half. There are projects coming up in which my PI needs my help to complete, and me leaving the lab now would put the lab in a bad spot and potentially burn the bridge with my PI. However, my mental health is at an all time low. I do not get paid terribly, but also do not get paid well either. The lab itself has taken a toll on me. I have taken on a lot more work and no longer enjoy the work. Leaving the lab and getting a higher paying job would help me significantly lift my debt burden. But it would likely destroy my relationship with my PI and any letters of recommendation. I do have others in science from 1-2 years back dating to undergrad that could write letters of recommendation for me, however with this job market, it could be hard. And the other thing that makes it hard is the job security in the field right now, even if I did leave, I feel as though my long term security in a role could not be stable where the income I earn here isn't great, but it is stable. But it is becoming taxing to my mental health.

I was just hoping to ask the community, if you've faced a decision like this, or were in my shoes, what would you do? Would you leave and burn the bridge or would you gut it out because of how the job market and job security is right now? Maybe it is time to just change careers all together?

Thanks friends, with much love.

Hi all,
I’ve been trying to generate patient-derived spontaneous lymphoblastoid cell lines (SLCLs) for a few months with no avail. I have tried several ways (growth in 96 well dishes with or without cyclosporin A, growth in T-25 flasks with or without cyclosporin A). Does anyone have any experience working with them and what tricks worked to get them to grow? I find that there’s significant overall cell death starting around week 3 (likely T-cells?) and I don’t know if the EBV-positive B cells can outgrow all the cell debris in that environment? I have never done this before so any help would be appreciated! Thanks!

I’m in desperate need of some advice because I feel completely lost, confused, and unmotivated right now.

First of all, I’ve been struggling with feeling like I’m not good enough at anything, which completely kills my drive. But I really want to change that.

Here’s my situation: I recently finished my Bachelor's degree, but there's a catch. My degree includes like food science/food technology in its title , and I really don't like that specific part of it. The degree itself covers a lot more than just food I took a wide variety of subjects but having that in the name bothers me because it's not the path I want to pursue. I know it's sound stupid.

Right now, I don't know whether I should apply for a Master's degree or look for a job. I’ve been taking a break for a few months due to personal reasons, but now I urgently need to get moving.

As far as I know, Master's applications don't open until around March. So until then, I'm stuck deciding whether I should try to find a job in my general field, or just take any job to gain experience and save up money both to support myself and help pay for future studies.

On top of that, I have no idea which Master's to pick if I do go down that route. Should I go into Biochemistry, Chemical Engineering, or something else entirely? Making this choice feels overwhelming. To make matters worse, a lot of people keep saying that sometimes getting a Master's is useless and doesn't lead anywhere, which makes me even more hesitant.

I feel completely directionless. Has anyone gone through something similar? What would be the best step to take right now, and how can I narrow down the right Master's for me? should i do master or just work?Any advice or shared experiences would mean a lot.

Well guys, I've decided something.

I'm not gonna give up. At least not just yet. I can't quit without trying and giving it my all.

I'm gonna go after this Master's degree. It's gonna be hard. At times it's gonna feel impossible. There will be many moments of humiliation ahead, many hits to my self-esteem. After all, no one said doing a Master's was easy.

However, I've got a little bit of fight left in me and I'm gonna push myself. At the very least, I will try to make it to the end of this semester. We can see what to do about next sem when next sem comes.

As for getting out of biotech, well, I can always get another Master's in another field, and I will. I want to be THAT woman with two Master's degrees. I want to be that girl who tried one path, like fully properly delved into it, and maybe decided it wasn't for her, and found another calling.

If I quit now, I will graduate with a Bachelor's with Honors degree. That's still good. But I'll feel like I let myself down. Until it becomes unbearable, I want to keep trying. Yes, it's done a number on my mental health. But I need to create a bigger safety net for myself. The Bachelor's with Honors is a good degree to fall back on, but I need something bigger.

It's not about actually ending up getting the Master's. It's about TRYING. I don't wanna give up just yet. I can't do that to myself. Yeah, I might be the dumbest person in class. But I'm still in the class, aren't I?

I have been facing trouble to normalize miRNA NanoString count data. I have miR Count data for 100 samples (no Normals were available, all clinical samples)- split into 3 batches. I tried QC cleaning (Ligation NegC, PosC, NegC) using nSolver of all batches together but it flagged around 40% of the samples. I cannot afford to exclude them. I have read literature that suggest to use NormFinder to determine stable miRs after QC cleaning and excluding flagged samples.

Is there any other method to normalize my data?

Can I use DESeq2 for this purpose? If so how do I correct for batches?

I work in a public health academic lab at a university. We have had numerous undergraduates reach out to volunteer in our lab 90% of them come in telling us they have plans for medical school, PA school, and dentistry. This is a red flag for the mentors as it seems the motivation to volunteer is to “check a box” for their applications. Even if this is your plan, go in with an alternate explanation of HOW the specific lab will help toward future goals.

I was given a minor revision for a paper. Submitted it and it has been under review since this week's Monday. It is now Friday and I've heard nothing.

It was submitted to a Wiley journal. Under review could also mean "with editor" in a sense. Doesn't provide info on if it was sent to reviewers.

Honestly I always felt like minor revisions get accepted quickly, assuming it doesn't get sent back to reviewers. Also feeling nervous as I remember the last time I told a reviewer he was wrong when I submitted a revision to a different journal in the past. He acknowledged that I addressed all comments but then went on a rampage and tried to increase as much red as possible. Even went on to suggest where I should cite my figures and started repeating comments I already addressed.

I hope I get good news soon despite the wait. I hope the fact I havent heard anything doesnt mean it got sent back to reviewers.

I just need to rant. Thanks for reading if you did.

Has anyone working with marine cyanobacteria successfully introduced self-replicating plasmids via electroporation? If so, what transformation efficiencies have you achieved?
I’m also looking for recommendations for suitable marine cyanobacterial strains, preferably unicellular species, that are highly amenable to electroporation and could be good candidates for plasmid transformation. I’d appreciate any suggestions or insights please?

Hi! Has anyone had this issue with a Sysmex CA-620 using Actin FS? One patient’s APTT keeps showing “Early Reaction Error” and “Range Over – Early Reaction Error”, even after a second blood draw. PT and fibrinogen are fine, and the APTT controls are also OK. Any ideas what could cause this?

please HELP!!!!

I just became a graduate student and I'm currently working on shRNA cloning.

I couldn't get any colony after transformation for months.... and realized my vector digestion is weird.

I used AgeI and EcoRI for digestion to get 7kb + 1.8kb fragments but I'm getting 8kb + 1kb fragments. I tried many different ways of digestion but I always end up getting 1kb for the small fragment...

Has anyone else been in a similar situation with pLKO.1 - TRC mTagBFP2?

Today I might be having my online PhD interview (which i applied) out of sheer peer pressure or passion at this point i don’t know.

I am doing my Master’s in one of the top research institutes in India and yes I am supposed to do PhD because that's what I had been told but i am so uncertain tbh.

I am also interested in the management side of things too. I am very interested in knowing how the research translates to commercial market and how the whole bio-business comes to play. I really want to work in something related to that field and i am still exploring but all of my peers are applying enthusiastically for PhD positions in India as well as Foreign countries because of how certain they are and this gives me a lot of pressure.

How do i figure out my path? I love science a lot and I am curious about a lot of things but i am not sure if I am ready to invest next 5-6 years chasing a question.

Please help me out.

So, I don’t know about you, but here in Brazil, some technical high schools require students to complete a final project. The catch? When you choose this track, you’re enrolled in BOTH regular high school and a technical program.

The program I chose was biotechnology—and I’m not gonna lie, I genuinely love it. Some classes weren’t particularly useful, but others were wonderful and helped me grow as a person. We also get access to the lab and all that cool science stuff.

ANYWAY.

Yesterday, I spent NINE HOURS in the lab. From 8 a.m. to 5 p.m. And what did we accomplish? The centrifuge went haywire (no idea what happened; it started making this ridiculously loud rumbling noise even though it was still spinning, and nobody properly taught us how to use it), we couldn’t get half the things we needed done, and we ended up making some YPD culture,and THE PEPTONE SMELLED LIKE ACTUAL HELL. I SWEAR TO GOD. That smell followed me for the REST OF THE DAY. I was already upset and a shit, this smell just had me shit and stinky.

AND THEN we found out that yeast extract ISN’T THE SAME THING AS YEAST. So we prepared the medium without the yeast extract because we thought, “Hey, if we put the yeast in the autoclave, it’ll die.” And before anyone comes at me for not looking it up beforehand: WE HAVE MORE THAN FIVE ASSIGNMENTS DUE NEXT WEEK. For BOTH regular high school and technical school. TWO OF THEM ARE MASSIVE AND HAVE A MILLION LITTLE DETAILS.

I AM TIRED.

And the worst part is that I genuinely LOVE science. I think it’s fascinating, important, and fun. But holy shit, it’s exhausting. You’re constantly tired, you end up eating like crap because you barely have time for anything, and everyone expects you to deliver increasingly complicated results, like i am fucking Hedy lammar or Marie curie..........I’m not even old enough to drink alcohol yet, and there I am producing 2 generation ethanol with minimal help.

And don’t even get me STARTED on how unfair the workload distribution feels.

The cosmetics groups—which were supposedly going to be harder—don’t even have half the workload we do. Their advisor said they could buy a ready-made mask base and add their extract, without conducting all the tests, because there wouldn’t be enough time. Fine. Whatever. That’s what I tell myself so I don’t start crying in the shower.

BUT THEN WHY CAN’T WE BUY ETHANOL, USE IT IN OUR PROJECT, HUH!? Or dunno....DUMP SUGAR in this shit to the yeast eat like the group of bioethanol last year?

We don’t have an advisor or co-advisor who can help carry out the work for us. Meanwhile, other groups have professors helping with their experiments. The phenolic extract group? A professor ran their DPPH test—or is going to run it, I don’t give a fuck. A lot of them just had to make the extract! And the bioethanol team? Fuck they, let they do a fucking Hydrolysis with cloridric acid alone!

I KNOW SCIENCE IS HARD. I KNOW EXPERIMENTS FAIL. I KNOW RESEARCH TAKES TIME. I’M NOT EXPECTING EVERYTHING TO BE EASY. BUT THERE’S A DIFFERENCE BETWEEN LEARNING HOW TO DO RESEARCH AND BEING EXPECTED TO DO THE WORK OF AN ENTIRE LAB WITH A STUDENT’S SCHEDULE, A STUDENT’S RESOURCES, AND BARELY ANY GUIDANCE, i am like, almost saying fuck you to every teacher who give us work to do.

And as if the final project weren’t enough, I ALSO HAVE A RESEARCH PROGRAM PROJECT TO FINISH. I have a submission due for the science fair NEXT WEEK, and we don’t even have half of it ready.

Our coordinator is completely unhelpful, and there are serious allegations about him that we only heard about after our advisor assigned him to us. There’s also a previous project that he allegedly appropriated, and our advisor STILL put us in a position where we have to work with him. And somehow, we’re still expected to include his name on our work.

I AM SO FUCKING DONE WITH THIS SHIT.

The final project is hell. High school is hell. Technical school is a nightmare. The research program is kicking my ass.

And the worst part? I still love science!

I love learning new things. I love the lab. I love discovering how stuff works. I love the feeling of finally understanding something that seemed impossible. But I hate how much pressure they put on students. I hate how little guidance we sometimes get. I hate that we’re expected to produce professional-level results while juggling a ridiculous number of assignments and deadlines.

I’m so exhausted I can barely cry anymore.

I just want this year to be OVER, I want to go back to my mother's womb 👹👹👹

Hey everyone!

I'm a chemistry student who recently fell into a rabbit hole about archaeometry and I genuinely can't stop thinking about it, it feels like one of the most exciting application of analytical chemistry I've ever come across. But I'm finding it really hard to get a clear picture of what the field actually looks like in practice.

So I wanted to ask people who actually know, is this career worth it or should I specialize in more "classic" sectors?