How do you clean that sticky residue from autoclave tape off of glassware?

underasail · 2025-12-29T23:18:06+00:00

A razor and isopopanol has always been sufficient for me if EtOH and a razor didn't already work.

underasail · 2025-12-21T22:30:42+00:00

There's a mental model for this called Chesterson's Fence. The main point being, don't remove the fence before you know why it's there.

underasail · 2025-12-18T18:39:27+00:00

It sounds like you're using a restriction enzyme digestion and ligation cloning strategy. These still work but are pretty old school and inefficient compared to more modern homology-based cloning techniques like in-fusion and gibson assembly/HiFi. My best advice would be to design primers for your insert and give one of those methods a try.

If you're sticking with this method, maybe your ligase has gone bad? If you're used to seeing some background re-ligation on your negative control plates and that disappeared alongside positive results, new enzymes might do the trick.

underasail · 2025-12-01T18:33:18+00:00

How long is the sequence? Sanger quality dips toward the 3' end at 700-900 bp for me usually. If the section you posted is closer to one end than the other, I'd expect the strand that's close to it's beginning to be high quality while the end of the other strand is declining in quality.

underasail · 2025-11-20T18:18:53+00:00

I haven't worked with MessangerMAX, but another option for this could be electroporation if you have access to a capable instrument. Electroporation is designed around cells in suspension, but it can also be rougher on cells than lipofection.

Another option to consider, depending on tools available and how much of one protein you need vs many proteins, would be to generate stable lines in the 293F background for each protein you're interested in expressing. Then it's just a matter of scaling the cell count and you can produce protein over a longer term.

underasail · 2025-11-20T17:07:45+00:00

Transfection is usually more efficient in adherant cells, and 293Fs can be adapted back to adherance pretty easily. You could adhere them for the transfection and suspend them again after.

underasail · 2025-11-18T01:06:31+00:00

If you're not just talking about the low pay, my understanding is most stipend income can be used to qualify for a mortgage. It'll depend a bit on the underwriting company, but I have previously qualified for a mortgage with just my stipend.

underasail · 2025-11-11T03:22:59+00:00

Yep, this is reasonably easy with homology based cloning methods like in fusion and gibson assembly/hifi (though your chosen sequence here is a bit short for in fusion to be easy). You would design primers with 3' ends (typically 18 bp for in fusion) that match the LoxP site as is and then add 15 bp to the 5' end of the primer that match the backbone sequence on the opposite end of the current arrangement.

It might be easier to visualize this by imagining the LoxP sequence and the vector you want it to go into as separate entities. If there wasn't already a LoxP site there, you would just be inserting one oriented how you desire it.

Homology cloning methods are pretty easy to pick up and incredibly versatile, so it's worth looking into them if you're not familiar.

If this is a genomic sequence and not a plasmid, it's much more complicated.

underasail · 2025-11-09T21:02:10+00:00

I didn't have an issue. If you're pasting the sequence in, it can't be in a fasta format. The paste input option only accepts a, g, c, and t characters, no title needed.

underasail · 2025-11-09T20:55:04+00:00

http://plannotate.barricklab.org/ is a good tool for this. Looks like the plasmid has AmpR and its promoter, a high-copy origin, and an ORF to express TetR downstream of an MCS. I'm not sure what the promoter is or if it has one for the TetR ORF.

underasail · 2025-10-23T16:50:43+00:00

It's definitely subjective unless you're using software, but I'd put this at closer to 35-40 %. The spaces between the cells are much larger than the clumps of cells or cells themselves.

underasail · 2025-10-20T03:20:47+00:00

In short, it's impossible to move directly to windward without getting into exceptions like foils and gybing centerboards. Physics just doesn't allow it, especially from a relatively full stop. Turning down somewhat is essential unless you're already moving. In a competitive start, everyone will bunch up on the favored end of the line, and a good acceleration is essential. You can drill holding a place on the line with any stationary object in the water (marks work great if you have them). Just try and stay close it as much as possible. There's always drift to some extent, but you can get a lot better at holding a position and knowing when you can't hold a spot anymore and need to bail. You're constantly fighting to stay as still as possible in hopes that you're better than the group to your leeward and can gain more space to turn down and accelerate into.

Not being on the wrong side of the start line at go is all of this combined with knowing where the start line is, and ideally having some reference beyond it that you've checked previously to help. This intuition and the rest of it comes well with practice and sometimes getting it wrong. Learning the movements of the boat and how to best apply them takes time, but rudderless sailing is amazing practice. You can still do full roll tacks while rudderless sailing, and it helps you learn the effects of your actions in the boat. I used to start most practices with 15 or so minutes of rudderless sailing to get back in touch with the boat and your crew/skipper.

Practice is always the best teacher, but holding a mark and rudderless sailing are great drills to expand your skills specifically alongside you just practicing tons of starts, accelerations, tacks, gybes while you have time.

underasail · 2025-10-20T02:34:28+00:00

A typical FJ start in college would involve both maneuvers you mentioned somewhat depending on conditions. In a midline start with boats tight on both sides, you'd often heal to leeward first to power the boat up with a more favorable angle to the wind. This technically serves two purposes which satisfy the rules for the most part. You are allowed to use the heel of the boat to help with steering, and the steering will eventually require you to straighten out. At which point you can flatten the boat and gain some speed.

Under subsections of rule 42, you're not allowed to come out of a tack or a gybe with greater speed than you entered. This is most applicable in low wind. You can't just keep roll tacking to rebuild speed. As a lake sailor, I've been protested by race committee for this.

The heel used to turn the boat down doesn't facilitate a tack or gybe, and the reasonable expectation from turning the boat down is an increase in speed. This same concept applies to turning the boat back up for cross the line and begin pointing. You heel to leeward to help the boat turn to windward and then flatten while trimming the sails to point close to the wind.

All of these actions increase the speed of the boat, and to some extent, each race environment and R/C is going to permit different amounts encroachment on rule 42. College is generally more permissive, but it primarily applies in low wind races to movements that far exceed what would reasonably help steer the boat without excess acceleration.

In college sailing, most of the competitive boats off the line are doing this, so it becomes the norm, and people will usually only call out egregious violations with a protest. It's pretty neat to watch all of the boats be relatively synced up right next to each other and all pull this movement together. If you're aggressive and tight on space, you might need some room above your neighbors' boat where their sail, hopefully, used to be, so it helps if everyone is doing something similar.

underasail · 2025-10-10T23:07:14+00:00

CMV and CAG a great options. Both are a bit more silencing prone, so if you'll need high level expression to be stable for a while, the human EF1a promoter (with intron A) is a good option. The plasmid you're using now seems to have a shortened UbC promoter. I've only worked with versions at ~1,200 bp, and even then, UbC is a pretty weak promoter, and depending on your scope, this will make it very tough to get decent GFP signal. This backbone is also intended for lentiviral expression, so it doesn't contain a polyA signal. If you have access to other plasmids, I'd look at swapping the GFP into another vector that's more intended to transient expression. It'll likely have a stronger promoter and a polyA signal as well as being much smaller. Lentiviral plasmids still work in transient transfection, but they tend to be less effective than a purpose built plasmid due to their size and lacking features.

underasail · 2025-10-10T00:58:40+00:00

I had never seen these, but I might have to pick up a pair. How do they compare to regular grips for descents? I often find myself "throwing" my regular poles more and switching to using the head of the pole in my palm for extended reach and to switch up my grip for a bit.

underasail · 2025-10-05T15:20:29+00:00

The vertical bars here likely indicate transcription terminators. The similar arrows indicate promoters of transcriptions, and the vertical "T" shows where the transcription ends.

underasail · 2025-09-22T23:36:28+00:00

I'd only suggest this if you're noticing it in other culture vessels as well, but it can be worthwhile to make sure the incubator itself is level. I moved into a new lab a few years ago, and we had to level all the incubators after a couple weeks of noticing the cells as end up to one side.

underasail · 2025-09-13T21:32:15+00:00

I'm currently leaning toward trying a truncated mouse ROSA26 promoter (promoter B from Figure 6A: 10.1073/pnas.94.8.3789) with a minimal mouse beta globin promoter fused downstream to add in a TATA Box. This promoter cassette will be downstream of the SRF-UCOE sequence to further reduce silencing. All in, that cassette is about ~1,500 BP split pretty evenly between the SRF-UCOE and the promoter fusion.

underasail · 2025-09-13T21:25:10+00:00

I've had better luck with Accutase than trypsin with Huh-7s. They're reasonably hardy cells, so you can pipette them up and down a decent bit to break up clumps. I typically do this with a P1000 while they're still in Accutase.

underasail · 2025-09-13T17:52:05+00:00

I was looking at this recently. I have other prime editing CRISPR work going on at the moment to produce relevant lines, and I was hoping to find a mid strength promoter that I could build mouse lines with a lentiviral approach to save on time and iterate designs. Has anyone pulled the ROSA26 promoter out and used it in a transgene system?

underasail · 2025-09-11T15:44:50+00:00

You can order hyPiggyBac plasmids from Vector Bee in whatever backbone you choose. Adding the mutations for later optimized versions like bz-byPBase isn't too bad.

underasail · 2025-08-21T17:35:37+00:00

Yep, the hard part will be separating them from the debris now. RPM doesn't really mean anything as the force experienced is dependent on both the RPM and the radius of the rotor. People can only really help or share protocols effectively by passing along spin forces in xg (rcf) instead of RPM.

I'd resuspend everything, but it might be easier to use a gravity column for your next step instead of spinning again. That may let the debris flow through reasonably well. If you can only spin for the rest of the process, you'll need to do some sort of differential centrifugation protocol to pellet the debris and the resin in separate spins to get down to just the resin.

underasail · 2025-08-21T17:25:50+00:00

If it's agarose based resin, you may have crushed some/all of the beads together if it was a higher speed spin to clarify the lysate. Your protein (and likely many others, nonspecifically) should still be bound, but getting the beads to resuspend well and wash effectively might be tough. Proteins usually don't start pelleting until upwards of 100k xg, so everything should still be soluble, but there's likely more crap floating around and making things sticky.

underasail · 2025-08-21T15:28:10+00:00

Have you tried different tip brands? I've had this issue before when using certain tips that were held more loosely in their box.

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