u/modestfish

Another possibility is a normal multichannel but transferring from tubes in a rack wherein the tube to tube spacing is about double the spacing between wells in a 96wp. You can just load tips on every other channel and transfer that way. I’ve recently been extracting DNA from filtered water and this trick really saved my life. It’s more cumbersome to keep the layout consistent, but it doesn’t require a specialty pipette.

https://preview.redd.it/3ttufjp0lsjh1.png?width=1052&format=png&auto=webp&s=c97d5a0ebe82cfc85e56a6cd4e5438a4218c5e68

For most suppliers of semaglutide--the kind that sell to compounding pharmacies like Hims or Noom--it's good ol' solid-phase peptide synthesis. It may seem like an elaborate compound at first glance, but it's prepared by very repetitive amide bond couplings of amino acids derived from natural sources and modified with protecting groups.

There are two components to semaglutide that distinguish it from run-of-the-mill peptides made from proteinogenic amino acids, however: the alpha-aminoisobutyric acid (Aib) in the second position of the peptide and the C18 fatty acid (with linker) attached to the sidechain of a lysine toward the middle of the sequence. The Aib, which is not one of the 20 amino acids typically found in proteins, can be added during the solid-phase synthesis steps. The C18 fatty acid component seems to be typically added during solid-phase peptide synthesis by using a protecting group on the lysine that can be removed without removing any of the other protecting groups on the peptide.

Brand-name Ozempic from Novo Nordisk is prepared by yeast fermentation followed by chemical modification, which Novo Nordisk will tell you is the only way to prepare semaglutide with as clean an impurity profile as the material subjected to clinical trials and therefore is the only one you should trust. The bulk of the compound is synthesized recombinantly by yeast fermentation. Relying on ribosomal synthesis for preparing a peptide comes with the disadvantage of not being able to (efficiently) incorporate amino acids outside the standard 20-amino acid repertoire, however. Chemists need to take the wheel to add the Aib amino acid and the lysine sidechain modifications. These two parts are coupled after the precursor peptide is isolated from the yeast fermentation, probably by coupling the linker+C18 fatty acid to the lysine first and then coupling the first two amino acids (histidine + Aib) in one reaction.

What about newer generations of GLP-1 receptor agonists, like tirzepatide and retatrutide? These two peptides include more non-natural amino acids in their sequences. Tirzepatide (Zepbound) has another Aib at the 13th position of the peptide. Retatrutide has another Aib at the 20th position of the peptide, and the non-natural amino acid alpha-methylleucine at the 13th position. With so many non-natural amino acids, it's not likely that a fermentation method would be economically feasible compared to the solid-phase synthesis approach, which can incorporate non-natural amino acids as easily as the classic 20.

These look like they are either different plasmid structures (supercoiled, etc) or plasmid multimers to me. I’d run a diagnostic restriction digest with an enzyme that cuts once on the sequence. You should see a single band afterward. Different plasmid forms will disappear since it’ll all but cut into linear dsDNA, and any multimers will be cut multiple times to yield the same single linear dsDNA.

Swirl is how I got started with R, so I think it’s a good way to begin! I really liked having my entry point being fully contained within R Studio; it encouraged me to focus on getting familiar with navigating the first IDE I ever used, and made it feel less daunting to extend my learning with the R For Data Science book once I got comfortable with the basics.

In case anyone returns to this post: I've found that these 1/8" silicone slip caps work really well to seal the outlet end of the nipple-tipped Sterivex filters. These particular caps are a little too long for the Sterivex outlet, though, so I cut them in half. They stay secure, even during shipping.

I always hated working with Fmoc amino acids; the solubility, the cis-trans isomerism of that giant kite of electrons causing chemical shifts of everything near it to shift like crazy…

Definitely dq strongly coupled to the protons in the adjacent signal—very clear second order effects here.

I've been a relatively happy emu user for analysis of Nanopore full-length 16S sequencing data, and judging by how often I seem to see it being used, I gather I'm not alone. The database is a little outdated, but it's not hard to use a custom 16S sequence database with emu.

While emu doesn't output ASV sequences, it seems that some authors have generated consensus sequences from reads that map to a taxon ID to generate ASVs that can be used in downstream analyses like PICRUSt2 or what have you. I'm not really sure that this approach makes sense, though; it can't account for intragenomic 16S heterogeneity, for instance, so you may end up generating a consensus sequence across two ~10% different 16S genes from a single species.

Given the reaction is taking place in ammonia, and given that the intermolecular product would yield such a large ring, I think the NH3 would be the nucleophile. I think it’d be a mix of ortho, meta, and para, given the similar propensity of benzyne formation at each site adjacent to the Cl.

Sane folks CAN'T submit to this journal--submissions are open only to members of their little gang! The "journal" is a barely-disguised blog.

Z-DNA is left-handed and has only a minor groove, similar to how it’s presented here. This is fine!

Literally just had this problem. Did a side-by-side vector-only control that gave about as many colonies as did the actual Gibson assemblies with NEBuilder. Colony PCR for all the negative controls gave no amplicon, and all the actual assemblies gave the intended band. Happy that it worked, not happy that I spent several hours on a Saturday night implementing an alternate cloning strategy before I thought to do a quick colony PCR before I chucked the plates.

Omega Biotek kits have performed very well for the price for me. They have really detailed size selection data for their mag bead kits too, made it a cinch to develop an NGS sample prep protocol.

Zymo has been a mixed bag. I like their Clean and Concentrator kits for PCR cleanups but I keep getting shit yield with their gel extraction kit.

Yep, mass specs were big issues in at least three separate circumstances I’ve been involved in. Ovens, freezers, and vacuum pumps (like those for mass specs) in particular have been problematic in my experience.

Be mindful of ventilation/cooling when trying to pack instruments into smaller spaces. With every lab move I’ve been involved in, the PI/management shoved instruments very close together, and the heat they generated being so packed would cause so many problems with them, not to mention the discomfort of working there when it got so hot. One lab would be consistently 80-90 degrees or so.

Electronics vs sterics

I’m still waiting for protein sequences to be available in the data warehouse. DNA sequences are there! Where are the amino acids?!

For some assays I’ve run, protein loss due to labware adsorption (on polypropylene) was really severe, like 90% due to the low concentration of protein being handled. Low-binding labware helped but didn’t fix the issue. 0.01% Triton X-100 did the trick though, regardless of labware used. It’s very protein-dependent, though. Some are totally happy super dilute in normal tubes for hours, other just will latch onto the labware in minutes. If whatever you’re doing has a little detergent (or BSA) you’re probably fine, but it couldn’t hurt to ask the vendor for some low-binding tube samples.

Former flavin-dependent halogenase enzymologist here--that step I'd just call "amine halogenation", with the product being some haloamine.

Is this an M1 MacBook Air, and do you have the Apple silicon (arm64) version of R installed? I’ve had issues with that R version in using some Bioconductor packages. You might wanna try installing the x86_64 version of R instead. It works fine for me performance-wise.

There’s a truly awful set of bike lanes near where I live—in Houston, on Weslayan St, that regularly two lanes of traffic traveling 40-45 mph. No cyclist ever uses them. Even without the cars, they’re half dirt and debris and barely wide enough for cycling even without the crud in the way. I’m certain few drivers on that road are even aware there are bike lines there since the paint is so faded. They’re totally pointless and serve only to put a green line on Google Maps on a street that doesn’t deserve it. Bike lanes like this need to be removed to prevent any hapless cyclist from getting the wrong idea that a bike lane means safe cycling.

Basix = F-tier

The expenses that were covered for us were just the cost of the movers. They did cover lodging for us as we attended the grad student recruitment weekend, but that was pretty much it.

Moving expenses were mostly reimbursed by the institution my PI moved to. We had to arrange for the movers ourselves, and expenses slightly exceeded the budget so we had to pay a little extra (a couple hundred each maybe). I didn’t receive a raise, and I don’t think anyone else did either.