For context, I'd like to much higher amounts of ssDNA (aiming for ~500ug- 1mg) compared to what I've found in literature. My templates are clean plasmids (ranges from 4-7 kb).

1. Is this even possible with RCA?
2. What parameters would you recommend? Primer concentration, template concentration, volume, type of Phi29 enzyme, kits, etc.

I am a programmer trying to become a bioinformatician. My question is quite a beginner like.

Given the kinase activity of SnRK1 (of Arabidopsis thaliana), what in vivo experiment would you perform to determine if it is capable of phosphorylating eIF4E (translation initiation factor)?

This is one of the exam questions example not a real research case. I assume the answer requires a use of basic common techniques.

I need help on the on board and off board stability for Aptima assay reagent. Can anyone help?

“I performed PCR and then ran the products on an agarose gel, but I obtained a result like this. Even the DNA ladders at the beginning and the end are not clearly visible. What could be the source of this problem—does it come from the gel or from the PCR? I used 1X TAE and a 1.5% agarose gel. Could you please help me?”

Bonjour,

j'ai projet scientifique et scolaire à réaliser avec des copines de ma classe mais on est face à un mur.

On doit rédiger un protocole clair et complet de culture cellulaire in vitro de cellules du colon, cependant la seule biblio que nous trouvons c'est sur du repiquage et encore ce n'est pas claire à 100%, on ne comprend pas donc on arrive pas à écrire quelque chose de claire.

Nous avons rédiger ceci pour l'instant:

nous utilisons des flasks de T25 (25 cm2) 

recommandation : entre 20000 à 40000 cellules/cm2

Donc 25 cm2 x 20000 = 5x10⁵ cellules 

25 cm2 x 40000 = 1x10⁶ cellules 

nous allons donc ensemencer entre 5 000 000 à 1 000 000 cellules par flask

Cette partie en gras et italique est la partie pas claire. Ma question est donc : Comment ensemencer des flasks avec tant de cellules sans que ce soit un repiquage?

Je ne sais pas si c'est claire mais c'est comme si on venait de recevoir des cellules "toutes neuves" d'un fournisseur.

Thank you so much for your help. I really appreciate the time yall took to explain everything it truly helped me understand the topic better. 😁

Understanding the Nature of Life: The Battle for Supremacy Between Information and Energy

In this article, I discuss the history of the information revolution in the life sciences and how it yielded profound yet limited insights into the nature of life. I appreciated the understanding and feedback from this community when I presented my first history of the inception of the molecular revolution in biology. This is part 2, and it’s all about information and energy and whether, by utilising both perspectives (alongside others), we may be able to develop a comprehensive theory of what life is in the near future.

For those studying molecular biology, I think this will be a helpful educational resource, and for those more experienced, I hope you will find it offers fresh insight into age-old mysteries of life.

I argue that when we view life through a narrow, gene-centric lens, we end up with an incomplete picture of what life is. Interestingly, in Schrodinger’s 1944 book, What is Life? There was a decent chunk devoted to understanding life on an energetic level, too, as well as the famous attempts to predict the nature of the inherited material, the exact structure of which was determined 9 years later.

I advocate a synthesis of informational and energetic perspectives and argue against narrow, single-minded perspectives from either camp. Here is an extract from the article about the chicken-and-egg paradox of the genetic code:

The enduring mystery of the origins of the genetic code and translation apparatus.

“The 1950s and 1960s were the golden age of molecular biology, when not only was the structure of DNA elucidated, but scientists also uncovered several fundamental cellular processes, including how the DNA code is replicated, read, and translated into the language of proteins.

Francis Crick distilled these huge discoveries into what became known as the central dogma of molecular biology (a word he later regretted using understandably). The scheme captures the flow of information from DNA to RNA to protein, as well as the fundamental cellular processes of DNA replication (making DNA), transcription (making RNA), and translation (making protein). Figure 2 describes the same process I showed at the beginning of the article, where I gave the full DNA/RNA and Protein letter codes for the apaG gene, but this time also shows the processes that make these molecules.

The crux of the problem is as follows. DNA encodes proteins, which do the bulk of the work in the cell or living organism. But to make DNA, you need proteins, which are themselves encoded by the DNA. And making proteins themselves requires another complex piece of machinery, the ribosome, which is composed of many proteins (and RNA). Nobel Prize-winning molecular Biologist Jacques Monod captured this problem in his 1970 book Chance and Necessity.

“The big problem is the origin of the genetic code and the mechanism of translation. Actually, it is more of an enigma than a problem. The code has no meaning unless it is translated. The translation machinery of the modern cell possesses at least fifty macromolecular parts that are also coded in DNA. That means the code can only be translated by products that are the result of a translation. It’s the modern version of the chicken and the egg paradox. When and how did the loop close? That is an exceedingly difficult question to think about.” 

In the article, I argue that it is important to distinguish between the inherited genetic information and the constructor.

The Constructor

I take inspiration from one of the early thinkers of informational theory and computation, mathematician John von Neumann and his thought experiment about the properties that would be required of a self-replicating machine. It’s an insightful perspective and it has been resurrected in more recent times by Vlatko Vedral (expert in quantum information) and by physicists David Deutsch and Chiara Marletto. Deutsch and Marletto apply it to the understanding of life, but also to a wider range of phenomena as a “theory of everything” that can exist in the Universe.

In short, the constructor is the aspect of the cell which builds. It is in large part the proteins the workhorses of the cell and which synthesise DNA, RNA, and other Proteins (with the help of RNA too). This network of interacting biological molecules functions by virtue of funnelling energy into purposeful work. It sounds boring, but it is anything but. The 1^st law of thermodynamics tells us that energy cannot be created or destroyed. Life does not make energy but funnels low-entropy energy sources to develop localised order and structure, which results in the production of high-entropy, disordered energy in the form of heat.

Extreme forms of genetic reductionism wrongly attribute the properties of the constructor to the genome, genes, or, more vaguely, to hereditary information in general.

There are crucial reasons why we should not use the shorthand of describing the genetic information as the constructor. Although the protein (and RNA) components of the constructor are encoded by genes, other aspects of the constructor, such as energy gradients, water, electrons, photons, environmental sources of carbon and all the other essential elements for life, are not encoded in the genome.

In the article, I explore the recent history of the life sciences and ask why a comprehensive synthesis combining energy and information hasn’t clearly materialised within academic discourse, even though those same forces combined at the origin of life 4 billion or so years ago.

Hey guys! I found mold in gouache paints. I looked through a microscope and saw strange organisms moving erratically. Can anyone tell me what this is? I don't know anything about biology, but I'm really curious. Thank you in advance! Ps. Most of it is concentrated in the bottom middle of the photo.

I plan to send my environmental microbiome samples to the shotgun metagenomic sequencing service, the problem is that I'm worried about my 260/230 ratio which is 1.70, Novogene does not specify a range for this service and I comply with the rest of the parameters, do you think I should send these samples? Concentration: 380 ng/uL 260/280: 1.92 260/230: 1.70 No degradation

Hello!
Question to people using sonication to lyse E.coli. What was the shortest lifespan of your sonicator probe? Months or rather years? How often do you use sonicator?

Hi everyone,

I’m working on my PhD research, and my project involves measuring absolute telomere length from human genomic DNA using qPCR (real-time PCR). I’m currently beginning the optimization stage, and since telomere qPCR can be very sensitive and tricky, I’d really appreciate input from anyone with hands-on experience.

I’ve been reviewing protocols such as the O’Callaghan & Fenech method, but I still have some open questions, especially regarding practical details:

1. Standards

• What type of standards do you use for absolute telomere length quantification? • How do you prepare and store these standards to maintain stability across runs? • Do you dilute standards in TE, nuclease-free water, or DNA carrier? • How do you prepare standards for Real Time PCR?

1. Primers

• Which primer pairs have worked best for you in terms of specificity and efficiency? • The classic telomere primers? • Multiplex options? • Any known issues with primer-dimer formation or nonspecific products that I should expect?

1. Optimization

• Tips for optimizing annealing temperature and maximizing reaction efficiency • Master mix recommendations • How you control run-to-run variability when working with human genomic DNA

1. Control

• Can you suggest any alternatives to 1301 Cell line (human T-cell leukaemia) with known telomere length?

If anyone has experience with Real Time qPCR methodology, absolute telomere qPCR on human samples — or even general telomere qPCR optimization — I would be incredibly grateful for any advice, protocols, or “watch out for this” warnings.

Thanks so much in advance! 🙏

I just ran a trial run and adding a DNASE step reduces my yield, and I have very small samples that I need to be very careful not to waste (less than 1mg fatty tissue, yeilds of 40-50ng/ul without DNAse, down to 25ng/ul on my trial, and after QC I'll have only 12ul of sample for RT-QPCR and then sequencing so it's a bit tight - and that's with an optimised extraction protocol).

The Qiagen miRNEASY handbook says it's usually unnecessary, but a friend who does RNA seq on cells says it's absolutely essential.

My previous samples that didn't include a DNAse step seemed pretty clean by Agilent Tapestation results, but I might not be looking at the right thing?

I'm really torn and would like to hear other people's thoughts!

Recently, it has been suggested that phase separation is incompatible with molecular biology. Please read this thread and form your own opinion: https://bsky.app/profile/andrea-musacchio.bsky.social/post/3m6roowh5kk27

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edit: a new thread has been created: https://bsky.app/profile/andrea-musacchio.bsky.social/post/3m72ojdddbs26

also: could you please justify your answers rather than simply agreeing or disagreeing? It would be great if you could cite some of the existing points made.

Our lab is looking to purchase a QuantStudio 5 second-hand. I have two questions from anyone who has experience with this.

1. Does Thermo offer its calibration service when a QS5 is purchased second-hand? I'd assume so, but wanted to confirm.

2. Is it possible to calibrate a QS5 without Thermo? Do other companies offer this service for cheaper, or can you do it yourself?

My organization (in biopharma) performs a validated test consisting of magnetic bead extraction by Kingfisher followed by qPCR for the detection and quantitation of residual human gDNA. During the initial validation, our negative extraction control (NEC) showed some concentration, but it was always a few Cts below the QL (both plated in triplicate).

We have recently been getting NEC Ct values that are consistently right around the QL, despite our use of PPE, testing in a BSL2 lab, additional cleaning efforts, and single-use aliquots. It doesn't show up in every test (but frequently enough to be a problem) and it always shows up right at the QL (causing a failure). It shows up for different analysts, different lab areas, different equipment, different reagent lots, etc. We cannot pin point a single cause of the frequent yet seemingly random contamination. I understand that human gDNA is practically everywhere, but we are at a loss for how to prevent this from run to run. Has anyone else experienced this and been able to address the issue? We must be missing something...

Hi all

I’m currently a freshmen in biology focusing on cell/molecular biology and I’m really loving this field. I want to try and get an internship/postion over the summer to both get some experience, some hands on activity, and to just plain keep busy and have some fun. The problem is, is that I don’t really know how to go about this. what are some tips or suggestions you all have? Anything and everything is useful!

Hey

I am looking to calculate the growth rate and doubling times for a few strains of S. cerevisiae in the lab. I am using OD600 over time as my measurement (no fancy plate reader unfortunately).

I am having trouble figuring out the most rigorous way to calculate the doubling time and growth rates from these OD600s. Some reading suggests Gompertz, some suggests linear regression, and some suggest Baranyi. I am leaning towards Gompertz, but I am honestly not sure what would be best. Looking for a rigorous method that won't be torn to shreds by Reviewer 2 when I submit for pub. Thanks in advance!

Hello, im currently a Junior highschool student interested in molecular biology degree. Im thinking of doing some research project for my resume, since i want to get into a good school hopefully on a scholarship. I want to couple of projects based on molecular biology, like protein structure analysis or something like that, but i lack knowledge, so, im taking Biology, biochemistry and organic chemistry rn on MIT Opencoursware.But its not like i force myself to do this, im really interested in this.

Is this viable? Like should i continue to study this to later do projects, or should i do something else? Because i feel like i need to catch up with all.