/r/labrats

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/r/labrats

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0

Two questions regarding transfection and confuency

  1. Is having enough cells for the experiment the only reason for doing transfection of cells (particularly HEK cells) at good level of confluency (usually around 70 %)?

  2. My second question is if I want my HEK cells reach 40 to sixty percent confluency the day after splitting, what should be the split ratio?

I did transfection at 25 percent confluency. I need only four cells. My transfection went well (I have lots of cells that are fluourescent), but still I am curious and looking forward to hear from you.

I have no word to thank you enough. Thanks for your answers in advance. I read posts in this group and everyone is amazing at helping others!

0 Comments
2024/03/28
14:36 UTC

0

Detecting encapsulated siRNA from nanoparticles

Have been working on some very seems to be trivial thing but everything failed. I need to know the recovery of siRNA after I encapsulated them in the NPs and isolated them from the reaction, and they are not the typical LNPs.

Some context is like I use NPs to encapsulated the siRNA and I use an amicon to concentrate the NPs. Spin down won't work as they stuck together and won't resuspend in water after 2 hr sonication.

I know the EE as it is 100% (nothing in the flow though, I dried it and looked at the fluorescence of the labeled siRNA), the problem is when I have to concentrate the NPs, some NPs stick to the amicon membrane.

After I recover the NPs, I have to use some method to take apart the NPs, but it is mostly heparin and TE.

For (low RNA concentration detection) ribo, I diluted the stuff for 100 times and still the matrix is affecting (rotational effect, in comparing std curve and standard addition) it is probably due to the heparin in the samples. But heparin and siRNA have similar size and I have no idea on how to remove them. siRNA is ~12.5-13.5kDa while heparin, according to online info is around 10-15kDa.

Fluorescence of NP/ taken apart siRNA won't work and I'm not 100% sure why. For "taken part", it is by doing a titration of heparin with same amount of NPs on a gel.

I have tried Ribogreen, gel (making siRNA std curve in a gel too lol), nanodrops. The recovery can be higher than 100% depending on what sample/method it is. No two methods agree with each other. Any thought or if you have good experience on this?

0 Comments
2024/03/28
14:27 UTC

1

US visa options - post doc interview

Hi lab rats!
I'm helping coordinate an upcoming visit for a post doc candidate in our academic research lab in the US. He is a current graduate student in Qatar and requires a visa to visit. I'm researching US nonimmigrant visa classifications and could use some help.

I understand if hired as post doc he will work under a J1 visa. However I'm not sure which visa classification is best for his candidate interview.
Any experience with these matters? TIA!

1 Comment
2024/03/28
14:17 UTC

1

How to handle epichlorohydrin safely

Bonjour All. Recently my PI asked me to synthesise new epoxy monomers from bio-based materials, i.e., diphenolic acid, using epichlorohydrin (ECH). From the literature, 1 equivalent of -OH requires 10 - 20 equivalent of ECH, meaning that I need to use 100-200 g of ECH for each synthesis. The problem is that ECH is toxic and cancerogenic. I understand that handling ECH should be careful. However, I don't know if I can handle or conduct the synthesis with that much ECH in a normal fume hood. For safety, do I need to work in a glovebox?

Many thanks.!

0 Comments
2024/03/28
14:11 UTC

1

Options for clinical rotation as CLS

Hello, so I am a health science major. I didn’t rotate in the lab for clinicals. I am now applying for my license. It’s required here in CA. I have a 10 year experience as an MLT when I was in the Navy. My question is will that suffice as my clinicals? If not, what are my options to work as CLS?

0 Comments
2024/03/28
14:06 UTC

5

How do I freeze cells without a Mr. Frosty container?

I’m waiting on an order but I need to freeze cells soon. How to freeze cells w/o those -1C containers? Are there any protocols online that show this?

17 Comments
2024/03/28
13:43 UTC

2

Helping international postdocs getting started in the US for the first time?

Hi Labrats!

I’m supervising an international postdoc who just moved from another country to US few days ago. I was wondering what might be some recommendations and timelines that I can help to assist them to be onboard as soon as possible?

The biggest challenge is that the postdoc has never been to the US before. They don’t drive but before they move to the US they got a driver’s license from the original county. (Need computer tests, road tests, and probably extra time for practice)

They are in the process of house hunting (currently lives in an air bnb). And they have more to do including driving tests, bank accounts, and buying groceries & house furnitures.

They currently commute by Uber because our institute location is in rural US. No public transportation.

For the above reasons I think they will be slower than the other postdocs started at the same time. I’m going to check out our postdoc office to see what assistance they can provide for sure. At the same time I’d like to ask if anyone has similar experience can let me know how long the whole process could take to settle down, and what else I can do here? The appointment is only 2 years so time is ticking but I also want them to be comfortable….

1 Comment
2024/03/28
13:26 UTC

1

Problems with vwr

Hello everyone,

I am comming from Central Europe and I want to ask: Are we the only ones who haves such major Delivery Problems with prepared growth Media from vwr and more specificly with with their supplier Reactivos. During the last 7 month we had barely any Delivery without any Problems. I am currently extremly unbeliebavble pissed

0 Comments
2024/03/28
11:36 UTC

13

Cute lil science logos - ideas for more please?

6 Comments
2024/03/28
10:38 UTC

1

Fighting with Primary Astrocyte culture (mouse)

TL;DR: No issue with neuronal cultures, as easy as ABC. I try to follow protocols for mixed cultures in order to then separate microglia/OPCs and Astrocytes, and everything dies.

I am honestly unsure what the fuck is the problem right now. I have tried following 2/3 protocols for this online, mainly this because it was the one used by whoever worked on this before me and I have their own summed up version of the protocol.

The real issue is that I am not even sure at what level lies the problem. The cultures seem clean a few days after making them, and while not as perfect as seen in picture, they are almost at 70% confluency by DIV8/9, which is when I am supposed to make the passaging to isolate astrocytes.
I am currently staring at "2 t75 flasks with isolated astrocytes derived from 1 t75 mixed culture" and there is barely anything that looks alive.

2 Comments
2024/03/28
10:36 UTC

1

Apoptotic cells

Hello everybody! I have a question here please enlighten me if you know, I searched a little bit on the internet, and couldn't find anything useful, so I thought any of you might know something. In my current experiment, I irradiate cells with alpha particles and X-ray daily for a week ( 5 fractions) and then look at some endpoints, I wonder if there is any effect from apoptotic cells or floating DNA fragments on remaining cells. Any explanation would be appreciated.

1 Comment
2024/03/28
09:13 UTC

5

How valuable is coding expertise in the workforce?

I'm currently finishing up my interview process for PhD programs in biochemistry or cell biology and was wondering how valuable/competitive knowing how to code (R, python, etc) makes you in your post-grad school aspirations? One program I am interviewing with offers formal training in this subject and I was curious how significant ones your ability to code or not code has impacted your job searches post-grad school.

3 Comments
2024/03/28
04:55 UTC

36

intern season is approaching, here's my advice

If you find yourself idle and want to make yourself useful by cleaning the lab, whatever you do, DO NOT touch anything that looks like a sample or reagent! If you accidentally throw out/lose something/disturb someone's assay, you are going to piss someone off. Also don't go and try to organize things! Even if the lab seems like a disaster to you, it's not your disaster to fix (yet).

What you can do instead:

  • Empty the biohazard bins/waste bins and make sure to put a new bag in the bin. Bonus points: fold a few empty bags and put them in the bottom of the bin, so the next person to empty the bin doesn't have to search for a bag.

  • Refill pipette tips, or bins of tubes, vials, caps, etc.

  • Make (or order) fresh bottles of commonly used reagents/buffers (don't throw out old ones though, you never know which one of your lab mates might be hoarding something for some reason) **only do this if you are 100% sure you know what you are doing!

  • everyone always is looking for lab markers, tape, pencils, timers... grab a beaker and toss all of these common items in it. Put it on the lab bench. Someone will be pleasantly surprised

  • clean glassware/dirty labware in the sink - be aware of what you are washing! Especially if you are in a chem lab.

Feel free to add to the list!

27 Comments
2024/03/28
04:25 UTC

43

Lost lab notebook. Need advice

Long story short, I took my lab notebook home with me to have all the details I needed to prepare for an oral presentation at a regional conference which was held in a different city.

The presentation was on Friday, and after that, I got lazy and decided to return the lab notebook on Monday (like.. why not have some fun in town instead of heading straight back to school? It's a dumb idea thinking of it now).

But, over the weekend, the car got broken into, and my belongings were gone, which means the lab notebook is also gone.

The issue is that when I told my P.I. that I lost the lab notebook, he got infuriated and accused me of theft because the lab notebook is technically his property. He stated that he will take this to Student Affairs to escalate this matter. And it looks like he already did, since I got an email from Student Affairs requesting a meeting to be held to discuss this issue.

I am not sure how to handle this matter. Advice would be appreciated!

60 Comments
2024/03/28
03:41 UTC

4

How long was your "response to reviewers"?

Let's see if we can find a record here. How long was your "response to reviewers" in term of number of reviewers, number of pages, figures, etc., and in what journal? Thank you!

3 Comments
2024/03/28
02:51 UTC

0

Luciferase assay problems

I'm trying to use a luciferase assay to measure transfection efficiency in my immune cells with firefly Luciferase. I'm a little confused about how to analyze this data - I have a negative control of untreated cells and also a background well of media + Luciferase buffer/luciferin (no cells). I've noticed that irrespective of the number of cells I work with, the background luminescence is always very similar to the untreated cells well. So basically every time I subtract the background from the untreated, I get 0 lol, or even very high negative values. My raw sample values are all much higher.

Theoretically I guess this makes sense because regular immune cells shouldn't be luminescent, and the background measurement is done to account for noise from the plate reader. But based on this, how do I normalize my data? My initial thought was to subtract background and normalized to the untreated but based on what I've read in literature level don't always subtract the background? I'm not sure how to troubleshoot this. Any help would be appreciated!

12 Comments
2024/03/28
02:13 UTC

2

Issues with cell adherence or contamination? iPSC-CMs

Here's a link to videos/photos of the cells in question. Would love to hear your thoughts on what's wrong with them.

I’m having issues with my ipsc-CMs dying and falling off the plate en mass. This started in February of this year and is oddly similar to the issues we had early last year. Those issues were gone by August, and there was no clear explanation of what happened. We use CHIR and IWR1 to modulate WNT signaling. We use B27- and B27+ in RPMI 1640 with L-glutamine. I’ve tried troubleshooting various aspects of the protocol with no luck. This protocol has worked on these cells in the past, so I’m not very suspicious of things like cell density and small molecule concentration. I also thawed new cells in case maybe the pluripotency of our ipscs had degraded, but there’s no difference between freshly thawed cells and older cells. Our ipscs appear to be perfectly fine until we put them in RPMI. They get randomly cloudy at various points throughout the differentiation process, meaning I will have a plate that is fine one day and cloudy the next. When the cells stick, they still don’t necessarily beat; they are not even close to as good as before February. I was worried about contamination, so I cultured some of the supernatant both in the incubator and on agar, and nothing grew. Additionally, my coworker did a pcr for mycoplasma, which came up negative. My PI and the lab manager are not worried about contamination. Everyone seems to have a different opinion on what is going wrong, but nothing seems to hit the nail on the head. Nothing changed between when they were working and when they stopped. Everything I’ve tried has no obvious effect. I am unsure of where to go from here. Apparently, the cells have always been working on and off in this lab. I might just need to ride the wave until they start working again. I would love to know if other labs also have issues with their ipsc-cms going through periods where they simply refuse to differentiate properly. I’m happy to answer more questions about the protocol and the troubleshooting that I’ve already tried.

3 Comments
2024/03/28
02:07 UTC

0

Am I accusing her or not?

I am a new graduate student in the lab and just started learning new stuff. So the other day I was having issues with a shared reagent, that I’ve never used before and know nothing about, and I panicked. So I went to the undergrad (who has been in this lab for 2 years) who prepared this reagent (her name was on it) and asked her that if she prepared it correctly or anything weird happened. The next day her mentor (who is a postdoc) brought this up to me and said that she told him it seemed like I was accusing her when I approached her the other day. I ended up apologizing and explaining and everything but I just wanna know, does it make people feel uncomfortable when I approach people with questions like these? Or she was just being overly sensitive? (I am an international student so English might be another factor)

22 Comments
2024/03/28
01:20 UTC

1

Collagen coated plate with suspension cells?

My lab only had collagen coated plates available and I am doing a proliferation bioassay using suspension cells.

Would the collagen interfere with the bioassay or should I be ok?

Let me know if you have any experience, thanks!

2 Comments
2024/03/28
00:40 UTC

13

Is ATCC currently... functional?

Recently I was struggling with my culture of HepG2. I posted here in the r/labrats sub and received some wonderful suggestions! After purchasing some collagen-coated tissue culture flasks and anti-anti, I ordered a new cryovial of HepG2 last week.

I know that ATCC does not ship anything on Fridays so as not to have perishable cells in transit over the weekend. I therefore strategically placed my order on Friday of last week, so that it would ship out on Monday, for delivery on Tuesday. I placed my order, and checked my dashboard to confirm that they "preparing my shipment," with an estimated ship date of 3/25.

Monday comes and goes. No shipment notification. No contact or alert of any delays.

Tuesday comes and goes. No shipment notification. No contact or alert of any delays.

Wednesday comes and goes. No shipment notification. I am aware that they have an 11:00 a.m. cutoff for shipments. After 11:00 a.m. passes and I see my dashboard unchanged, I call ATCC's one and only phone number. The recorded voice tells me that there are six people ahead of me. I spend 40 minutes on hold waiting to speak to a human being, and the queue decreases by two people. Finally I can hold no longer.

I've seen past posts from years ago that there were problems at ATCC. Is anyone aware whether these problems are resurfacing? I thought this was supposed to be a reputable non-profit -- and effectively, the largest repository of cells and other biologics in the world. HepG2 isn't exactly an exotic cell line -- I'm wondering what could be causing the delay, and also impacting customer service for those answering the phones.

9 Comments
2024/03/28
00:14 UTC

1

How appropriate is it to report a mean and negligent TA?

Hi all,

Undergraduate senior here currently enrolled in a Gen Chem 2 lab (not a chem major though, I’m an ecology student). Yeah, I know, senior year is late to take a chem 2 lab, but I went all the way through organic and analytical before my advisors told me I was supposed to take the lab for Gen chem 2. Kinda interesting.

Anyway, to cut to the point, my TA is absolutely atrocious. He is genuinely mean and aggressive towards students. If you make any mistake at all or do not understand what he is saying, he will roll his eyes at you and raise his voice. He will treat you like you are the stupidest person alive for accidentally making a mistake. He’ll roll his eyes, say. Words however, cannot do justice to the way it makes some of us feel. It genuinely makes my blood boil every time he confronts me. I’ve been through multiple biology and chemistry labs now and I have never, EVER had anyone remotely CLOSE in just pure ugliness to this man.

In addition to the constant downgrading and ridicule you get as a student, he rushes you and will treat you like a prisoner who isn’t cleaning the floor fast enough. I am a very cautious person in the lab - in fact, I have severe Obsessive Compulsive Disorder which makes me even more cautious, and even more slow. I’ve started becoming careless and rushed with my chemical usage because I am so terrified of upsetting him. It genuinely makes me lose my mind, because I can’t decide to be safe or please his desire to leave.

Fast forward to today. I was OK up until today. I kind of just figured I would deal with it and move on. We had our lab - qualitative inorganic analysis. I spilled a tiny bit of lead (II) chloride solution out of my test tube. I immediately told him and I was told to simply wipe it up with a hand towel. The towel that I was provided to dry my hands off with after washing my hands. Needless to say I didn’t feel safe with that outcome, I dried my hands on my jeans after thoroughly washing them. We have a spill kit, but he was too lazy to use it. Instead of washing my test tube’s residual stubborn precipitate content out thoroughly (which includes lead chloride, silver chloride, and Copper Ferrocyanide), I was told to simply dump it down the sink. Seems like an aquatic hazard, to say the least.

In conclusion: he treats everybody in the lab horribly, like we are all dumb and a massive inconvenience to him. He rushes us to the point where I feel reckless and unsafe with chemical usage. He stretches the limit with chemical cleanup and cuts corners. Everything he does goes against what the lab coordinator teaches about safety, which is taken very seriously. I found the coordinator after class and told her that I got lead chloride on my notebook. I was told to rip my pages out of my notebook that were contaminated and wash up thoroughly. A completely different response from the TA.

I understand that being a TA is stressful. I understand that you have a time limit for the teaching lab. But we are students. We are bound to make mistakes. And stress is not an excuse for poor safety and leadership. I’ve worked with far more dangerous chemicals under hoods, and have ran out of time plenty of times. But those TAs always were very kind and respectful. They supported me and taught me. Nobody ever has treated me like this in my academic journey. I feel totally disrespected, unsafe, and negligent. It feels like I’m going to boot camp every class.

I have a good relationship with the lab coordinator. She is incredibly sweet and nice. Today I walked out of lab just angry and anxious about the whole experience. Is it appropriate to report this TA to the coordinator? Or is it inappropriate for some reason?

My apologies for the rant. Thanks for any help.

8 Comments
2024/03/28
00:01 UTC

51

how do you deal with under-qualified workers in the lab?

i work as a Scientist in Industry for high throughput production at a start-up. our manager hired a graveyard shift lab technician who claims he also currently works at an oncology lab (how he has time for this? i have no idea). i, like my colleagues, expected him to be proficient in pipetting. however, it’s clear that he is greatly under-qualified in this area. he has made various pipetting errors (e. g., accidentally adding more elution media than needed, cross-contaminating samples by reusing tips. one of my colleagues caught him working with chemicals without gloves on!!) that have greatly complicated our downstream processing. he has done pipetting training twice up to this point. i don’t know how else i can help him improve his pipetting skills. i don’t have the time to sit with him and point out every error he’s making while we’re in production; that’s what the two pipetting training sessions were for.

to those who may have had a similar experience, how did you proceed once you noticed someone is not performing well? this is my first time being in this situation and i’m completely clueless on how to move forward.

edit: i was typing this in a fast fury because i was irritated and needed to get it out. now that ive calmed down a bit, i thought i should mention a couple things that i left out. we had this lab tech do two training sessions for pipetting; once when he first arrived, and the second time when i initially escalated my concerns to the manager. i see the comments of people saying to talk to the manager (thank you for your responses!). when i first brought this issue up, my manager said to wait and “see if he makes any other mistakes.” he came off very… dismissive. my worry is that no action will be taken on this technician even after i escalate my concerns for the second time. i have no authority to fire staff… if i could, i would.

26 Comments
2024/03/27
23:07 UTC

0

Tips for sending DNA for sequencing to USA + Opinion on eurofins USA

Hi,

I am currently running some experiments in Mexico where I need to send plasmid DNA for sequencing to eurofins USA using the SimpleSeq Premixed kit. Does anyone have experience sending DNA to USA? I would like to know if you have any tips on how to package/label the samples in order to reduce the risk of it being delayed or stopped in customs.

At the same time, I would like to know your opinion on Eurofins USA regarding the quality of their sanger sequencing. I have read comments stating that Eurofins USA isn’t as good as the european version. I have used the european version of the SimpleSeq kit in Eurofins germany before with no issues, but I would like to know if anyone’s had any bad experiences such as failed sequencing with Eurofins USA.

4 Comments
2024/03/27
22:29 UTC

0

Any journal suggestions for a manuscript submission?

I will be submitting a diabetic neuropathy review paper involving some therapies. Any decent journal recommendations?

0 Comments
2024/03/27
22:04 UTC

0

What's wrong with my gel?

Help out a rookie please! These are gel electrophoresis results after running a gradient PCR to determine the optimum annealing temp for primers. Why the long streaks, what possibly could have gone wrong with the samples?

5 Comments
2024/03/27
21:56 UTC

2

Storing Individual Tissue Sections Long-Term

My lab is currently sectioning mouse brains for a transcriptomics experiment. However, when sectioning the brains, only one half of the sections will be immediately mounted on slides. The other half of these sections will need to be preserved in a “loose” fashion, in some kind of unmounted way, at -80C. Months from now, we will have to retrieve these individual unmounted sections from the freezer, thaw them out, and only then mount onto slides. Our sections will be 20um thick. My questions is whether this can be done, and if so, how.

This was my first guess: after taking these sections, we could gently place them in a well plate with OCT-filled wells. After making sure they were immersed by OCT, we could transfer them to the -80C freezer. When it came time to revisit them, they would thaw at room temperature for mounting.

Any other suggestions would be welcome. Thank you in advance

2 Comments
2024/03/27
21:26 UTC

2

IVT Producing RNAs too large? I am stumped

So I have been struggling with this problem for over a month now and have not been able to find a solution. Basically, when I run in vitro transcription to generate my RNAs (ranging from 50 - 100 nt) and then load the samples on a Urea PAGE gel (8% PA, 7 M urea), it appears that instead of my reactions producing single bands they are all producing multiple to many bands of increasing size. I have verified that the lowest size bands are almost always the correct-sized RNA, and so the rest are RNAs that are migrating slower for whatever reason (larger size? not fully denatured?).

I have use PAGE-gel-purified DNA templates to ensure clean DNA preps (DNA templates are PCR products), replaced all of the reagents for making my gels and loading buffers with brand new reagents (new urea, 10x TBE, new 40% PA 19:1), used a brand new IVT kit (just bought Transcriptaid t7 high yield transcription kit), played around with sample denaturation conditions (e.g., 85C for 2', 95C for 5'), used different stains (ethidium bromide and sybr green II, never stain for more than a few minutes). Nothing changes the results.

I have attached an example gel to this post (lane 1 = rxn without the RNAP, lane 2 = with the RNAP, lane 3 = with the RNAP + DNase 1)

Has anyone else had this problem? I am dumbfounded and in desperate need of a solution so any help that any of y'all can provide is much appreciated!

https://preview.redd.it/u6pe83k12yqc1.png?width=386&format=png&auto=webp&s=b9e9dd0bfb5dc28e2c0308070d10b56cc685ff0a

7 Comments
2024/03/27
21:22 UTC

1

PCR troubleshooting

Hi lab rats! My labmat ran a PCR gel and this was the readout. 3 samples appear to be stuck inside their wells — it’s like they haven’t migrated. This PCR product has an expected length of 800bp.

Any ideas or theories, even far-fetched, on what might have happened would be super helpful!! Thanks!!

8 Comments
2024/03/27
21:02 UTC

1

Genewiz/Azenta Amplicon-EZ

Is anyone here using Genewiz/Azenta’s Amplicon-EZ NGS service? The read data we’re getting back has truly atrocious mapping to our target amplicon (~20%) and appears to be full of reads from other submitters’ libraries. They BLAST to organisms our lab doesn’t use, and include reads that were clearly reverse-transcribed from spliced mRNA and metagenomics samples. They’re not environmental. Genewiz is trying to claim the problem is my sample, but I have no trouble sequencing in-house (we need to send this one out for QC SOP reasons). Has anyone else experienced this?

2 Comments
2024/03/27
21:01 UTC

1

How do I find this study?

The documentation for a particular amoxicillin capsule, it is stated:

In a multi-generation reproduction study in rats, no impairment of fertility or other adverse reproductive effects were seen at doses up to 500 mg/kg (approximately 2 times the 3 g human dose based on body surface area).

I've seen this statement in slightly varied formulations in various documents for amoxicillin products, but I've never been able to find the actual study. Is there some FDA or other resource where I might find it? Is it possible the results of this study haven't been published?

2 Comments
2024/03/27
20:56 UTC

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