I went back to grad school after years working government research and a decade of classroom work. Long story short, I am conditioned to remained poised when dealing with difficult people. I am older than my classmates and closer in age to my professors.

I asked a question relevant to our topic material and professor responded in a very nasty, mocking and condescending way. I calmly told her that she was being unprofessional for treating her students like that. She responded by telling me how much funding she has and how many publications she has. Remaining poised, I firmly stated that her accomplishments are wonderful but do not give her a pass to be rude to her students. In a classroom setting her students need to feel comfortable asking questions, and what might seem obvious to her may not be so for someone who does not have her level of expertise. She appeared surprised and continued with the lesson as if I never said anything.

Post below :)

I am inoculating 10 ml of bacterial culture overnight for plasmid prep using qiagen miniprep kit. The rest of the protocol i am following what the kit mentions. In addition to this, I am using NFW heated to 65 for elution, and i am standing the column for 5 mins.

After this, particularly with low copy plasmids, I am getting very less concentration (60-90 ng/ul) and 260/280 ratio is coming between 2-2.2. The elution volume is 50 ul.

I was following the same protocol earlier too, but facing issues since few days..

Please help..

I am currently investigating on the interaction between some amino acids of a protein with its substrate, and I want to test out by changing the initial amino acid to others that have different binding properties. I found the mmCIF model for that molecule, however I don't know any app or website to read mmCIF file or edit it like I want. So I just want to know if there is any way I can edit it to my idea like above?

Thanks so much.

hi! 3rd year micro/immuno PhD student in the US. I've been having some issues with my PhD lately (well, before the holidays) and feeling a lot of resentment but I'm not sure if the problem is just me.

I'm gonna keep the background info and general lamenting to a minimum. but the basic issue is my bosses (i am co mentored but one primary PI) feel I am not producing enough data despite me working a not-insignificant amount. one of my bosses has been really quite pushy about this and I think he is trying. to help. but it's kind of over the top and I feel like some things are potentially odd.

is it normal for PIs to text you late at night or on weekends asking for data? additionally, is it normal for a PI to be unhappy with a student for an experiment not working (or that experiment requiring troubleshooting)? is it normal for one graduate student to manage a large mouse colony with multiple strains (we have ~300 mice) alone with no help? is it normal to suggest or imply a student should be working nights and weekends (one boss will say it's okay not to, the other will go on long rants about how we should "want to" and "sometimes it's necessary")? is it typical for graduate students to work 12-14 hour days, often 6-7 days a week (i don't do this anymore, i can't keep up with it and be well physically, but many in the lab do). is it normal for drinking alcohol in the lab and while working to be encouraged? are "mandatory" "lab family" outings outside of work hours normal?? sometimes the outings are fun but i already work SO MUCH.

I have been working on essentially the same experiment for over a month, but when I ask my boss for help he just says he doesn't know or insists I must be making dumb mistakes/technical errors. he gave me a protocol to use which I have followed religiously numerous times and followed all of the advice he or my other mentor did actually provide but he frequently changes what he told me to do after the fact. I don't think it's intentional, but if the variable we decided to change at our meeting was volume of culture and after the experiment fails he says "well you should have done XYZ instead" i don't know what im meant to do... it's obviously rude to point out he's changing his words. they also expect us to be on call - if I don't reply to texts or emails after hours i get scolded.

i can't help but wonder if this is normal and I am not getting scolded and I am just sensitive and this is all just academic science working environment though. maybe I'm just lazy. my boss says I'm lazy a lot. but idk if he's joking!

sorry for the long post... and thank you to anyone who takes the time to answer.

Casting a wide net for a host of new equipment and have been recommended MP for a range of products (XRD/XRF like the Empyrean/Aeris and the mastersizer laser diffraction equipment). Are they worth the premium vs Rigaku/Bruker/Fisher etc?

Hello everyone,

I am trying to PCR amplify a DNA fragment (purple) using a plasmid as the template. However, I am encountering an issue with a significant amount of residual plasmid template remaining after the PCR. This poses a problem since I plan to use the amplified product for Gibson Assembly, and any leftover template plasmid could result in high background during transformation.

I have already tried diluting the plasmid template to 25 ng and 50 ng per 50 µL PCR reaction, but these attempts did not yield the desired amplicons, and the residual template plasmid was still visible on the gel. I also did gradient PCR and tried to use PCR enchancers (BSA, DMSO, Betaine and a combination of all).

Are there any strategies to minimize the residual plasmid template during the PCR step? Or is my only option to treat the PCR mixture with DpnI to digest the template plasmid and then gel extract the fragments of the desired size?

Edit: i use phusion polymerase from thermofischer scientific, i just ran the pcr again diluting the plasmid template in up to 1000x dilution which is 0.3ng of plasmid/PCR reaction - got no desired amplicons.

Hey everyone,

I’m new to bioinformatics (just started grad school!), and I’m going to working with huge datasets like whole genome sequencing, RNA-Seq, and transcriptomics. My current laptop is struggling, so I need to upgrade, but I’m not sure what to get.

Here are the options I’m looking at:

1. MacBook Pro with M2 Pro (24GB RAM, 512GB SSD)
2. MacBook Pro with M4 chip (16GB RAM, 512GB SSD)
3. MacBook Air with M3 (16GB RAM, 512GB SSD)
4. Maybe a high-performance Intel/AMD laptop? (No idea which one though).

I’ll be running memory-heavy tools (Python, R, Docker, etc.) and working with big files, so I need something powerful. I’m leaning toward the M2 Pro because it has more RAM (24GB), but I’ve heard the M4 is faster. Then there’s the M3 MacBook Air—would it be good enough with 16GB RAM, or is it too lightweight for bioinformatics work?

Also, the storage (512GB SSD) might be small, but I could always use an external SSD if that’s the best option.

Since I’m just starting out, I’d love advice from anyone with experience—especially if you’ve done bioinformatics on a Mac or another setup. What’s the best choice for someone like me?

Thanks so much for helping out a newbie!

Apparently most vendors have discontinued the surgifoam/gelfoam (also dental foam I think?) for soaking up blood during stereotaxic surgery. Has anyone bought some recently, and can you share catalog info?

Hello everyone, this is my first post and english it is not my first language sorry if my wording off.

We have some issues with our ultracentrifuge model Sorval MTX 150 from thermofisher, where between 01:30 and 02:00 hours of running it stops and shows the error in the picture. We have try everything that is recommended in the instruction manual: wipe off the moisture, cleaning the the door packing and even checking the whole vacuum system.

The error Always happens between that running time, never before nor after.

Has anyone encountered this error before? If so, what did you do to get it fixed?

Thank you all in advance

Hello!!

What is your best advice for a new lab manager? What are some mistakes you made and leaned from? Any advise for supervising technicians?

A GE Superdex 200 Increase 5/150 GL column of our lab was just found cracked and fractured (not the resin inside, the actual glass column body). We asked around, but nobody's admitted to mishandling it yet. We'd want to rule out the possibility of it happening spontaneously before going through the CCTV footage and pointing fingers. Has anyone known a column body cracking for no apparent reason (maybe room temperature dropping or internal pressure)? Many thanks!

We submitted a manuscript to a food engineering journal a month ago. Four reveiwers commented and with some revisions, it seemed that we had a good shot. My PI submitted the revised one, and out of nowhere we had comments from fifth reviewer. Understandably my PI was pissed and passive aggressively responded that " due to the unexpected requests of revisions, we will be submitting by Jan 10th 2025".

Is a surprise reviewer normal?

Hi Everyone,

I am a new PhD student (just had my first committee!!!). I am able to digest publications in my field, for the most part, but struggle with retaining this information. My PI, on the other hand, can recite bits of knowledge from papers that are relevant to our discussions and I want to start working to get to that level. It just seems like when I read a paper and understand it, I just can’t remember it. It sounds a bit naive I know, but I was hoping someone had any tips and tricks they use to fully ingrain the material into their memory cores. I am actively marking up the publications as I read but are there any apps that you use or flash cards to help with this?

Thank you!

I'm a fresh grad (gen/mb) and i've been looking for jobs on linkedin but ive got no success. i'm gonna do my MSc after giving my entrance exams next year in Feb. what job role should i apply for and should i apply though the HR or just message the company profile itself? what should i mention in my resume? please help, im going crazy sitting at home and unemployed.

Hey everyone, posting to see if anyone has been through this and can share experience. We had our sliding glass cooler overheat and reach 33 C, for no more than 48 hours. All of our reagents - mTeSR+ (basal medium, not complete), DMEM, additives, got warmed. We transferred them to our cold room (only place with enough room for all) as soon as we noticed and they're cooling back down. I feel like the basal medias should be okay since the important things like bFGF and VEGF are in the supplements in our -20. Also things like DMEM get warmed to 37 and put But other things like Accutase, MEM-NEAA, B-ME, Versene, I'm not sure about. We also have some specific products from STEMCELL, like CellAdhere Dilution Buffer, and specific basal medias, and finding information about these products is near impossible... Has anyone had this happen, and which reagents did you end up tossing? TIA!

There had better bloody well not be DNA in my PCR consumables, Starlab.

Hey, I used a proofreading polymerase to amplify a 5kb segment from the genomic DNA of some cell lines. The PCR took several optimizing steps and because of that I have run out of the purified gDNA. Due to this I was wondering if I can reamplify the product using the product as a template and using the same primers.

Couple of tidbits regarding the product:

Like I said it is about 5 kb long.

I ran it out on a gel and it is super clean. No other bands or anything like that.

I performed a PCR Cleanup on it and got back about 90 ng/µL.

I could re-extract the gDNA from the cell lines, but these cell lines were from a collaborator and would take a while to reacquire.

I need the whole amplicon so nested PCR does not seem feasible.

I saw that reamplifying PCR products can be a bit tough due to how concentrated they are. To approach this, I figured I'd use fairly dilute amounts in my PCR reaction. I was thinking of using 1-10 ng total in a 50 µL reaction. Is this too much? Is this too little? Some people say to also run it with a reduced number of cycles...I normally do 40 and was thinking about changing it to 20? Does anyone have experience with reamplifying products and can offer advice?

I have to make transport media for skin swabs, but my lab don't have sodium thioglycolate? Can you tell any alternative for this or do you know any other transport media I can make without it, Ames and Staurt both used sodium thioglycolate.

I have a question on suspension cultures, which I have never used before until recently. More recently, I tried thawing a new batch but when I followed the instructions (eg adding correct volume of expansion medium slowly drop by drop, centrifuge 300 g for 3 min etc) as closely as I could, I didn't see any pellet forming at the bottom. Furthermore, I put them in a T75 since the instructions said so, but I realised the flask may be more for the adherent culture, and my lab does not really use ULA T75 flasks. However, I've tried thawing and growing another suspension cell line before using the same type of T25/75 and it was fine.

It's been a few days, but I don't really see anything much except a few possible floating single cells (which is nothing like what the cell line datasheet showed as an example photo). Is this still salvageable if I wait a few more days? I'm also wondering if I should still try with trypsin, in case there are somehow some attached cells, and transfer whatever may be inside to a 10 cm ULA plate? The thing is, this batch is a newly bought batch, so I was extremely confused when I did not see anything.

Does anyone work with the amino acid solution for Empower? I'm interested in the methods and reports and as to whether they are anything special worth thr cost?