Hi

I don’t have a clue how restriction digestion works.

I want to insert a fragment into a vector. My manager suggested to cut the vector with two enzymes and then using neb tool to build a plasmid.

Finally i want to assemble all fragments via Gibson.

I assume that i design primers regularly where the enzyme cuts with overlaps for all 3 fragments?

Do i need to also cut the fragment out of the plasmid or pcr amplification of it should be sufficient?

Thanks

What do you guys think about the journal "Archives of Biochemistry and Biophysics"? Does anyone here have any experience publishing with this journal? Thanks!

Details: I'm trying to load palmitic acid to cda1 monomer for a CyTOF experiment. But for this to work, i need to mix palmitic acid and cda1 at a molar ratio of 40:1(PA:cda1).

Step 1. First i calculated the moles of PA. Moles= Mass(g)/MM =0.001 g /256.48 Moles = 0.0000039 moles

**First, i convertes the concentration from milligrams per milliliter (mg/mL) to grams per milliliter (g/mL) since i have 1mL of PA at a concentration of 1mg/mL.

Step 2. I then calculated the moles of cd1a

Moles= Mass(g)/MM =0.002gg /49000 g/mol Moles = 0.000000041 moles

**i converted the concentration from milligrams per milliliter (mg/mL) to grams per milliliter (g/mL) since my cda1 monomer is at 2mg/mL concentration in 1mL. ** Since cd1a is 49kDa i changed that to g/mol being 49,000 g/mol.

---- Help ---- I now want to to make a total volume of 200ul containing PA And CD1a at a molar ratio of 40:1(PA:CD1a). How to calculate this?

Also is my moles calculationa correct?

Thank you everyone

Hello, posting for my labmate whose account is too new to post:

"Hello, I have some questions about transient transfection. The plasmid used in my experiment is pcDNA3.1-FLAG-gene. I tranfected plasmids with the same sequence of insert but amplified from different colonies into 293T cells using the same transfection assay. After 24 hours, I collected the cell lysate and performed western blot. However, the protein expression level between them is not identical. What could be causing the difference?"

any help appreciated, thanks <3

Has anyone had any issues logging into their Benchling workbook using google accounts recently? Every time I try logging in it says to "try signing in again".

Got my bio degree a few months ago, and currently have been taking one post-bac class. I’d like to apply for a lab tech or research assistant job, but I don’t know if I could get one that accommodates my class schedule. I’m looking at tech jobs at the university I’m taking a class in and was wondering if there’s any chance if universities in general would accommodate schedules for an employee to work, go to class for an hour, and then come back to work.

Edit: Also asking in regards to research assistant positions as well.

I'm working with some imaging data of the spine at the moment and I'd like to see if I can identify any structures by looking at a transverse slice of the same part of the spine from either a dissection or imaging.

I will start by saying I have this job because my previous boss went in, talked to the PI, and advocated for me. This is a major reason why I don’t want to leave - I don’t want to disappoint my former mentor.

I also love the work itself but the lab culture is kind of negative. I wouldn’t go so far as to call it toxic, but the person supervising me is very stressed out by this task, and I feel her frustration pretty much every day. It makes me feel like they were fine handling stuff without my help. I was hired into a position that was supposed to take workload off of everyone in the lab but I don’t know that any of the grad students or research associates are happy about this change.

I’ve been here a month now and I am at a place in my training where they have invested a lot of time in me but there is still so far to go before I’m independent. Part of me wants to leave now (meaning as soon as I have another job lined up) before they waste any more time on me.

I just feel guilty and think maybe if I stick it out and finish training things will feel better. But then again I would rather work somewhere I feel valued. I have quite a bit of experience as a lab tech and come from a lab where I was given a lot of respect and responsibility, so it is weird to feel like such an annoyance here.

I think I’m going to talk to my former mentor tomorrow and see what he thinks but I’m wondering if anyone here has thoughts??

I ordered a couple of antibodies on Thursday and I received a delivery notification on Friday but nothing showed up. The delivery supervisor called me back to let me know they it was delivered somewhere else and has been kept at room temperature since Friday. Before making another order, I wanted to see if it is worth reaching out to UPS. What do you think

I just say "I work in a lab" because saying that I "conduct bacterial reverse mutation assays to detect genotoxicants in the early drug development process" is too much

Hi! I’m an undergrad in a genetics lab conducting research on a species of fish. I am the only person in the lab that has been able to amplify Chromosome 1 for differential expression, any ideas on a fun poster I could create signifying I am the only one to do so? Lol my PI said I should go for it, we have several funny posters it would go near.

Hello everyone. I am making a series of 10 short videos, where I break down a research article, explain why they're worth a read, and suggest related works. My focus is on articles published in the last 25 years (2000-present) in the life sciences/biological sciences. The target audiences are mostly university graduate students and PhD students in their initial phase. Can you guys please suggest me a few of your favorite articles?

Here are the criteria-

Field- I apologize for my limited expertise, but I’d prefer to analyze a paper from any discipline of life sciences/Biological sciences.

Interesting- Do you think a college student with their limited exposure to the research field would find it captivating?

Impactful- Not necessarily groundbreaking, but has a meaningful takeaway. Not something as specific as “protein X is one of the key regulators of Y pathway to propagate disease Z in organism T”.

Simple- Considering the length of each video will be 3-4 mins, I’d like to avoid articles with overwhelming data.  

Reason- Why do you like this paper? Can you please share the reason in a couple of sentences? You will be duly acknowledged through the video.

I appreciate any help you can provide and welcome feedback on the project. Thanks!

Edit- I want to show my students why someone find a random work of science interesting, not to ask them to read the abstract of a great paper.

Friends - help! I need to ship liquid-filled, 35mm tissue culture dishes (fixed adherent cells) cross-country.

How would one go about this? Do I seal the dish with something? Do I put the dish in a larger container filled with liquid? I’m at my wit’s end!

I recently graduated from college with a b.s. in biology last year and started working at this company for 11 months now. The division I work for is very small and it consists of 3 people, me included. I’m the only lab technician. Ever since last year, the company have been struggling financially, slowly taking our benefits away. They haven’t hired additional people the entire time. As I’m the only lab technician I get all the work. I’m so beyond stressed out. I don’t even get paid enough for what I do.

It’s so difficult to find another job in biotech. Should I talk to my a manager? Ask for a raise? Find a new job? Any advice help.

Pure precision in the lab: Nailed it with 40.0000 grams of PBS powder. The other lab techs said to share it here, so here it is!

I need to purify a 130 kDa protein from a crude plant extract. I have a very good monoclonal antibody and used it to prepare a imuno affinity column.

After column purification, I can see a lot of smaller bands present on the Western, ranging from 75 kDa to 20 kDa. Apparently these smaller bands are fragments of my protein. The intact protein (130 kDa) corresponds to 10% of what's on the lane.

Is there an easier way to get rid of these smaller fragments rather than using a size exclusion column?

Any suggestions are welcome.

I need help. I got laid off my pharmaceutical research position almost 6 months ago and I’m still struggling to find work. I have a molecular biology ASCP certification and experience with qPCR, Illumina NGS, cell culture, and DNA/RNA extraction. Do I need to go to grad school at this point or are there positions available that I’m just not finding?

Hi all,

So recently I have started work in a lab for my dissertation. From the research background I am coming from, I haven't had much proper research-related experience be it experience dealing with a lot of equipment to proper independent work. Coming to a lab with facilities above and beyond, I am truly overwhelmed by everything and I am losing major confidence, making silly errors due to my lack of presence of mind, and struggling with lab maths, to top it off, I have a graded assessment that will be marked based on how I perform and I can't help but think about all the little blunders I am doing which will affect this mark, and it's all been mentally taxing on me to the point that I am not sure if I have the confidence to finish this successfully, but at the same time, I also like what I am doing, and I'd rather d-word than live without a goal/my passion. Can someone suggest some helpful tips/advice on how I can overcome these mental challenges?

I froze and pulverized my mushrooms with liquid nitrogen (as per related literature that did the same) then sent it for DNA extraction and PCR but they couldn't find any sequences:( sent it twice now.

Any ideas why?

I accepted an offer from a PI and am not sure how to politely tell the other PIs II have been in contact with. I originally had meetings planned with them and I just feel like it is a very awkward email to write.

What are some things to avoid or include to be as respectful as possible?

Hey lab rats! I'm having an issue with my Thermo Fisher –80 Revco RLE series (aren't we all smdh) that I've not been able to find any discussion of on this or other subreddits. Basically, the display works fine until you try to get it to display the temperature chart, at which point the display fritzes for a second, then crashes and restarts. If you don't try to get it to display a chart it works fine. It's like the processing power needed to display the chart makes it fall down go boom.

It feels similar to an issue that u/snorksnek posted about eight months ago: https://www.reddit.com/r/labrats/comments/15xg432/anyone_ever_had_this_issue_with_their_80_we_cant/ except that following their guidelines of disconnecting the backup battery, turning off the breaker, unplugging the freezer, waiting 30 seconds, and plugging everything back and turning it on again didn't resolve the issue.

I popped off the bezel and unscrewed the screws holding in the display to check all the connections and they all seemed solid back there.

Do any of you magical lab fairies have any other thoughts before I drop more than two grand on a service call?

Hey y'all,

I've ran qPCRs only for gDNA and cDNA, but never for plasmids. I'm designing a qPCR experiment that involves a plasmid, what are some differences that I have to be weary about? Is it just easier to run a restriction enzyme digest to make the plasmid DNA linear?

Thanks in advance :)

Hello! Third year biotech undergrad at a large public uni in NJ. I was offered 2 internships after some desperate applying.

1 is in a large cosmetic company. 22/hr for 10 weeks, M-Th, 5 mi from where I'm located. The internship is in R&D but deals with batching and stuff like that.

2 is in a small biotech company. No pay only credits, 40 hr/week, 35 mi from where I'm located. Research is in cancer and it seems like a good stepping stone into pharma.

I feel like the pros and cons are so vast. idk what to go for. need some advice as to what I should pick. Thanks!

Hi everyone,

I would to know if someone here use a microplane reader especially the Cerillo Stratus and could help me please to use it ? The Manual isn't clear for me and I have difficulties with the calibration... I have send an email to te team of Cerillo but it's urge for me... Thank you in advance !

Hello! First time poster, long time lurker.

I am currently rethinking my career and was hoping to get some perspective from people out of my current field. I work at a biotech as a clinical lab tech right now. I have a bachelor’s degree in cellular, molecular, and developmental biology.

I am considering going back to school to get more into the research side of things. I did work as an undergraduate researcher in college for 3 years, rotating labs to see what I liked. I worked with zebra fish, plants, and fruit flies. I understand the importance of animal testing in research, but I personally really struggle with doing it myself. This has been the biggest hurdle for me when considering going back to school to pursue research. I worry I won’t be able to advance my career in research and be trusted to test on precious specimens like induced pluripotent stem cells without animal research experience.

I guess my main questions are:

1. Could I get away with going back to school for a masters in biology and avoid performing animal testing?
2. What would a career look like without animal testing with a masters in biology? Is my only option plant models?

I’m willing to accept that it’s not for me, but thought I would ask around first. Please let me know your experiences and perspectives :) Thank you!

Title pretty much.

I am helping with a new build cGMP lab with waived CLIA cert and ISO 14644-1 cert that has a ISO7 cleanroom and does minimal manipulation of biologics. Very small lab, maybe 300-400 sqft in an office setting.

I am looking for a good LIMS for this setup and wanted to know if anyone had advice on which one is the best. I've only ever used excel, and EfileCabinet or a clinical lab LIS.

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Any advice is appreciated!

I made it into the final round for a PhD position at a very well regarded Bioprocess engineering Institute.

Next week, I am suppossed to visit the lab to hold a short presentation about my life and scientific career and meet the rest of the team. I am currently in the process of preparing the presentation, but honestly struggling a bit with it, as I have a few projects that are not exactly scientific projects. Would you include things like iGEM, assistant positions and my stint away from academia or should I keep it strictly to my scientific work ( aka Bachelor and Master thesis?)

Is there anythingelse, that I should expect/ be preprared for?

If you got any recommendation on how to make the best out of the meeting I would be happy to hear them.

​

So, I found a used Tuttnauer Autoclave (model 5075ELP-B/L-D) on a resale auction site for laboratory equipment. The autoclave was built in 2022, and in the photos the LCD screen works. The bidding starts at $2,000. A brand new autoclave of this size sells for around $45,000 minimum so this is definitely a steal if we get it for anything under $10k.

My question is this: would you bid on a used bench top autoclave? Does anyone have any honest reviews of Heidolph Tuttnauer bench top series autoclaves?

P.s. this would be used in a QC microbiology lab at a pharmaceutical manufacturing facility to sterilize a bioburden membrane filtration manifold as well as coupons for cleaning validations, and other small pieces of equipment. We are currently purchasing all of our media pre-prepared. Thanks so much!!