Dear All.

I hope you are well.

I work in a lab from a couple of years now, I noticed one thing.

We have about 20 pcr cabinet in different labs (model uvp pcr2 cabinet mostly with air circulation option), Regardless of where their position is, they all have a peculiar smell.

I cannot tell the smells but it remind me smell of old plastic (like the smell of old masking tape) or brittle masking tape left under sun (brittle masking tape if touched release a kind of fine powder debria).

In addition , I had to sanitize some of them for an engineer visit, i notice that if I use a dump cloth to wipe the inside, the cloth become yellow. As if there is dust settled on the inner surface of the base and the walls.

if there is such thing , what I am concerned about is the operator being exposed to small particulate, maybe produced from plastic degradation of tips box , vial plastic bag or other plastic container left inside while the uv decon cycle is on.

Does anyone experienced the same thing ?

What's an interesting operon or another type of regulatory mechanism you know of?

Some weird or not well-known way that organisms regulate their genes and/or protein and RNA production. Or some viral mechanism like the phage lytic/lysogenic switch.

Hello Reddit hive mind,

I have the following problem: I want to isolate mRNA from medaka eggs. To do this, I have used and adapted the classic TRIzol protocol so that I have a more or less constant total RNA yield of around 360 ng/ul.

Now I want to isolate the mRNA. I expect about 10% of the total RNA to be mRNA. For purification I use magnetic beads with a poly-T linker attached. When I run 10-15 ul of my total RNA through the column, basically everything is lost and I can't tell why. (For reference, the sample starting at 360 ng/ul yielded about 0.2 ng/ul; I was expecting about 36 ng/ul).

Now I ran another sample (160 ng/ul) through the MACS, but skipped the pre-elution step as it should be discarded. My yield was 1.8 ng/ul, which is good. But the A260/A280 measurement was 0.8, which tells me that my sample is extremely contaminated (I expected it to be at least 2.0 or higher).

I am now considering running 100 ul of RNAse-free water through the column before eluting the mRNA with the buffer, as it is possible that the contamination is caused by the wash buffer.

Any help would be appreciated. If you have a similar experience, please let me know. I'm kind of helpless.

i use this protocol for the mRNA purification and start at step 1.2: https://static.miltenyibiotec.com/asset/150655405641/document_b9fauorr0d301eb29fag68252g?content-disposition=inline

Looking for experts in the US on CHRNA4. Have a nonsense mutation in exon 5, TM2 region, that is effing up my family’s (and my) life.

Am in the US. Don’t see that there are many or any people researching this. Would like to talk to someone who can comment on the effect of the mutation on protein structure so I can brainstorm therapeutic possibilities with my provider to prevent degeneration. If you know of anyone, let me know. I have read all publicly available studies but am thinking there might be more out there behind paywalls, etc.

I am not a scientist but am highly educated, so would like to consider based on the impact of the effect on the protein what type of therapies I might try.

TIA.

Hi all,

I’m a first year molecular biology/bioengineering & biochem PhD student at a well-respected research institution. While I know it’s common to feel imposter syndrome in this environment, I’m really struggling to overcome it and actually stop feeling like a fraud. My background before entering this program was a general Biology and Environmental Science degree from a small liberal arts. I did really well in my classes, but only participated in ecological research (nothing at the bench). I then worked for 3yrs in industry at a well-known synthetic biology company (this was at the bench). I was well-liked at the company, and was a reliable worker, but I feel like I am still lightyears behind the other admitted students in my program. I feel like I don’t remember too much detail from my undergrad courses and even forget a lot of basic molecular biology. I’m not really sure what I could do, or if I should even take a year off to review concepts/take courses to brush up? I feel like my admission to this program was a mistake and it was simply just a good GPA and a well-known name of a biotech that got me in when I’m not qualified. I’m wondering if anyone else has felt like this?

Hi all,

I'm an antiquarian who still uses EtBr as a DNA stain, but my UV transilluminator has died. It appears all I can buy nowadays is a blue light transilluminator. Does anyone know if this would still work with ethidium? MUST I update to GelRed (which is basically the same thing with a cute long backbone)?

To give context: I'm a third year Undergrad in Molecular Biology. This year we have a chance to apply for lab work (unpaid internship) as part of the studies. We have to apply with either mentors, institutes or anywhere that would have us basically.

I am currently in the proces of writing a short resume for applying because it's always required but since we aren't incredibly experienced in lab work (we have been in contact with most techniques and did them ourselves, but a lot of time it is group work or similar so I wouldn't say that there is a huge confidence with lab work) I am having a hard time writing it without it feeling dishonest (because my skillset isn't that advanced).

Does anyone have any tips/ideas for the content of the resume? Would it be better to focus on different skills apart from molecular biology methods?

Hello.

I'm an international student currently doing an MSc in Biomedical & Molecular Biology Research in the UK, and I need advice on finding opportunities to develop my research skills. Basically, I want to develop my laboratory skills in biochemistry/molecular biology/cellular biology as well as my research/data analysis skills. I'm not terrible at those things; I just want to spend more hours developing them outside of university hours so that I can be more prepared for my future career.

However, I'm only allowed to work part-time based on my visa, and I have been REALLY struggling with finding a single opportunity to help me improve my skills. I've looked for part-time jobs, internships, and volunteering positions. I haven't found a single position that I'm either eligible for (most require years of experience or full-time working hours) or that would help me improve the aforementioned skills. I've spent hours looking on job boards, university websites, volunteering websites, etc. and come up completely short. I've even used help services from my university and been unable to reach anything.

So, I need tips/advice on where I should look. What kinds of positions are the easiest to find? When during the year will those positions be the easiest to find (e.g. are summer internships my best bet?) What kinds of positions will help me develop the skills I mentioned?

If I'm chasing a lost cause due to me being an international student who can only work part-time, then please let me know. Would it be more advisable, for instance, to just wait until I complete my MSc and PhD before going for a full-time position? Or, is there some opportunity out there that can help me develop my laboratory and research skills from now on?

Thank you.

Hi all, I'm currently in my third year of a biology degree, and I'm taking molecular biology I. I feel like everyone else in my course is getting the material so fast, but I just can't get it. Memorizing content is one thing, but for me it's that I don't understand the concepts well enough to apply them to a diverse range of situations... I just genuinely don't understand how this stuff makes sense to people. If you are someone it makes sense to, good for you, and please send help LOL

I am a BTech Biotechnology graduate from India and would like some information on the Masters in Molecular Biology program which is combinely offered at Johannes Kelper University, Linz and Paris Lordon University Salzburg. I am interested in the program but would like to understand how the classes are held. The description states that in the third and fourth semester classes are held in both campuses. Do they expect students to commute between the two universities daily? I hope to find some alumini who could help me out with the details.

Hi!

I am trying to amplify the foxo gene in drosophila but am struggling to find the primer pair that works. I looked through several papers but had no success in finding sequences.

The cDNA works (checked via actin).

Has anyone done this and succeeded? If so, could you share the primer pair used?

I’m a molecular biologist with experience in working in different labs that miss traveling and want to try the life as a digital nomad. I’m not sure how to get there at the moment as I have a broad background in molecular biology.

My work experience has included analyzing microbiology/ virus and water samples. During the pandemic I worked with Covid samples and followed the new mutations. I have also worked as an embryologist and created embryos at the lab. When I worked as an embryologist I was responsible for creating the yearly report that included statistic data for our treatments as well as being a part of a team that worked with IT procurement.

At the moment I’m working with NGS detecting known mutations associated with different types of cancer. At my current job I do both the lab work and analyzing the data.

I have studied extra courses in python and was a part of a project during my thesis where I detected antibiotics resistance through building a database with known mutations.

Are there any digital nomads that have a similar background as me? And what do you work with?

At the moment I’m not sure what kind of jobs to apply for with my background.

Love and peace to this community

I posted some questions (below) on primase and RNA primers in DNA replication.

My reading has now begun to answer some of my questions.

The primase RNA primer history is tortuous and complicated.

The history of the discovery of DNAP by Arthur Krugman from 1956 to 1957 explains this complex and long path.

In 1956-1957, Kornberg discovered DNAP, which later became DNAP 1.

In 1953, Watson and Crick postulated that there might be an enzyme that catalyzes DNA replication. The method of DNA replication was still unclear. So Kornberg set sail on an uncharted sea.

Meselson and Stahl did their semiconservative DNA replication experiment only in 1958.

Kornberg discovered a DNA synthesizing enzyme by growing E. coli extracts with radioactively labeled dNTPs in a test tube. Incorporation of the radioactive dNTP took place but at a meager rate. The product was sensitive to DNase. Its synthesis also required the presence of all four nucleotides, A, T, G, and C and Mg.

Kornberg thus disproved vitalism, but he then set out to concentrate and purify the enzyme, which he had successfully demonstrated. He later grew DNA with a purified DNA template.

He thus discovered DNA polymerase. Like Columbus, however, he did not find the Western Hemisphere but came upon Haiti.

We now know that DNAP 1 is the most common of the five different E. coli DNAPs.

But it is not the processive one. It does not elongate DNA. In his initial experiments, Kornberg noticed that his DNAP synthesized DNA but also broke it down.

Kornberg won the Nobel Prize in 1959, even before Watson, Crick, and Wilkins did for describing DNA structure in 1962.

Kornberg did not discover the processive DNAP, which does the majority of DNA replication.

Cairns demonstrated in 1969 that E. coli mutants could grow and replicate without DNAP 1.

Kornberg won the Nobel Prize for discovering the wrong DNAP enzyme. I remember someone telling me that around that time.

His son, Thomas Kornberg, discovered the processive (elongating) DNAP (now III) in 1970. He did so in his senior year at Columbia University. The son saved the father.

I knew Thomas Kornberg at Columbia. He was also enrolled at Julliard to study the cello. At the time, Julliard was across the street from Columbia. Double enrollment was forbidden at the time at Columbia, but Thomas carried his cello to Columbia classes. I once asked how he avoided getting into trouble for his double enrollment.

Columbia now has a very prestigious double program with Julliard.

Thomas is now a professor at UCSF. But he still plays the cello.

www.youtube.com/watch?v=81rk7_I4-zY

Thank you for reading this.

Bohdan

BrooklynMD

My original post:

In almost all cases, DNA Polymerase needs a primer to start replication. The DNA Primase is a relative to RNA and DNA polymerase.

How and why does the DNA Primase stop so DNAP can continue? The RNA Polymerase does not need a primer, but it often starts and ends with short replicated aborted transcriptions! Are there biochemical parallels between the DNA Primer and the RNA Polymerase in this regard? Is the DNA Primer more closely related to the RNAP than the DNA Polymerase?

In the nucleoplasm, NTPs are much more common than dNTPs! Is that a determining factor that enables or demands the need for a DNA primase? Could this be a kinetic-determined necessity?

Is the DNA Primer use of NTPs a remnant of the RNA World?

I am reading genetics to understand RNA in preparation for a book on HIV-1/AIDS and NYC.

I am a physician and my many questions! :)

Thank you for reading this.

Bohdan

Those of you who do immunnohistochemistry, could you take a look at the following AB staining protocol and tell me if it makes sense? I've never done IHC before and neither has my lab, and I patched together a few protocols I found from former labs & the internet. I'll stain PFA fixed brain slices (40um thick) and I'll test out a few different concentrations for the primary AB. Here's the protocol:

Blocking mix: 2% BSA in PBS with 0.025% (2.5ul/10ml) Triton X-100 and 0.02% azide (2ul/10ml)

Day 1:

1.     Wash 3x10 min in PBS plus 0.025% (2.5ul/10ml) Triton X-100, shaker at RT

2.     Incubate in blocking mix for 1-2 hr at RT

3.     Primary AB diluted in blocking solution (1:x), incubate overnight

Day 2:

1.     Wash 3x10 min in PBS with 0.025% (2.5ul/10ml) Triton X-100, shaker at RT

2.     Incubate in secondary antibody for 2 hrs in the dark at RT in 1% BSA, 1/500 of secondary antibody in PBS

3.     Wash 3x10 min in PBS with 0.025% (2.5ul/10ml) Triton X-100, shaker at RT

4.     Counterstain by adding 5ug/ml DAPI in PBS at RT for 5 mins 

5.     Wash 3x10 min in PBS with 0.025% (2.5ul/10ml) Triton X-100, shaker at RT

Is there any advantage of using Tween instead of Triton-X, or TBS instead of PBS? This is not a super important experiment so I'd like to keep it simple. But please let me know if there is something obvious that I've missed or if it looks ok! Thank you!

I'm in a cell biology course and phosphorylation happens a lot. Why is the phosphoryl group so special? What does it do on the molecular level?

Hey guys I am working on recombineering for genome editing recently. I got a plasmid that carries donor DNA, but I am not able to tell if it targets the leading strand or the lagging strand on the genome. I know that leading strand is synthesized from 5' to 3' and the lagging strand is synthesized from 3' to 5', but it is still very confusing. How can you tell the direction of replication fork if you are given a sequence on the genome and how can u tell if donor DNA targets the leading/lagging strand if you are given a plasmid map?

hi all! i know in a typical golden gate assembly, your type IIS RE digested pieces do not have the recognition sites (i.e., you should design your pieces in such a way that the enzyme cuts out the insert, leaving the recognition site on the unused piece). however, i have a plasmid with BsaI sites whose backbone i want, and the insert i do not want. so in this case, i want the piece with the recognition sites on it - the piece you typically don't use. so my question is would my assembly still work if i am attempting to use this piece? my understanding is that as long as my insert has the correct overhangs this should work, and the BsaI recognition sites would be conserved in the final plasmid. thanks for your help!

Has anyone taken an online masters program for molec? I am looking to further my education but cannot do it full time. I’ve looked up quite a few schools so far but was curious if anyone has any recommendations?

I have plasmid that expresses a 20 kd protein-when I do the protein purification I get products at 20,40,60,80 kd. Is this plasmid concatenation? my loading buffer has a reducing agent (this should not just be disulfide linkages)

Hi everyone! Sorry this is so long! I’m looking for some thoughts on how best to apply my molecular biology and cloning skills in a way that is beneficial to my employer when our expression construct / plasmid cloning is being outsourced more and more. I have been doing protein expression cloning for ~18 years and knew it would someday be primarily outsourced. I do know how to do various types of protein expression but that is being done in a different department (in a location I don’t want to move to). I would love to find both a digital way to become useful and apply my molecular biology knowledge but also still use my lab equipment. I have been trained on protein purification via AKTA and could probably do more of that but, I admit, it’s not my first choice for where I would like to pivot. Most of my molecular cloning is to support structural biology. We have a whole lab full of nice equipment for molecular biology and I would hate to get rid of it. We have an extensive QC and databasing process for generated plasmids that takes a lot of time so one thought I had is to learn Python to help streamline that process. Another thought I had, to still use our lab equipment, was to shift to using the lab to troubleshoot anything that doesn’t express to try to lower the incidence of having to re-design constructs.

Anyone else dealing with this sort of transition in their careers or witnessed this change at a large company and have thoughts on skills that can be added for job security and / or new ways to use the lab equipment? Another thing I should note: luckily, though a pretty large company, my employer is very flexible and encouraging when it comes to employees learning new skills and taking risks with new technology. This is why I feel hopeful about making this work :). Thanks!

Eterna is a participatory science game run by the Das Lab at Stanford. We currently have 40 puzzles for solving the secondary structure of pseudoknots with different levels of validation (PDB down to completely novel). The puzzles are open to anyone around the world to play (homework break!): https://eternagame.org/labs/13612324

New players first solve 30 short tutorial puzzles before entering the "lab projects". All sequences submitted are tested by chemical mapping and the SHAPE data is returned to players in the game.

https://preview.redd.it/ridzo0m3opwd1.png?width=559&format=png&auto=webp&s=23780f47101770012c003832e39f725c9a5ca2ee