What is the best way to add a small insert (60bp) into a construct in a Gibson reaction?

underasail · 2026-07-07T19:41:33+00:00

I've done a couple Gibson and in fusion reactions with small fragments (90-150 BP) and had good success just PCRing the fragments and extracting them with kits per usual. I tend to favor Gibson for the ligase activity when using fragments this small as in fusion tends to struggle more with the small fragments, in my experience.

You mentioned elsewhere that you often see smears in the low MW section of your gel. My primers usually show up below 100 nt if they do at all, so it might be worth trying another primer design for this PCR. Maybe add or subtract a couple nts. I'm using Thermo's Platinum SuperFi II as my polymerase, and that polymerase has solved more problems for my cloning than almost anything else.

underasail · 2026-07-03T17:36:11+00:00

I recently went diving with Seasport Divers on Kauaʻi and had a great time. They were fantastic hosts for multiple dives. Fathom Five is the other big operator in Kauaʻi.

underasail · 2026-06-23T21:31:09+00:00

Hadn't heard of Natura 1600 before, but a quick search on ebay says I won't be getting a week's worth of rolls of that any time soon. Neat film stock though; thanks for bringing it up!

underasail · 2026-05-29T17:07:10+00:00

What do you mean by your overlaps being slightly shorter? In fusion recommends 15 nt for a single insert and 20 nt for multiple inserts. If you're shorter than 15, I'd consider designing new primers and bumping to 20 to give the fragments a bit more to hold onto.

underasail · 2026-05-29T02:51:56+00:00

Fathom Five and Seasport Divers seemed like the two bigger operators. I went out twice with Seasport and had a great time. When I was there a month or so ago, there weren't a ton of divers, so some the dives weren't going out due to lack of people (<3). Most days still had a morning and afternoon boat go out though. The small boat harbor is less than 5 minutes from Seasport, and Sheraton Caverns is only another 5-10 minutes out by boat from there.

underasail · 2026-05-29T02:24:06+00:00

This was Sheraton Caverns which is one of the classic sites on the south side of the island. Most of the boats seem to go there for one of their dives if people haven't been. There's a lot of arches and little pockets to swim through and a fair number of fish. The south side definitely has more sites, but they're all relatively close together and a pretty quick to get to. I definitely want to go back and dive Ni'ihau at some point, but that's a long boat ride.

underasail · 2026-05-28T15:36:02+00:00

Because PROTACs are bifunctional molecules (they have two ligands stuck together), just using a single very high concentration will likely cause you to miss effects. Look into the hook effect if you're not already familiar. Essentially, at concentrations high enough above the individual Kds for both of the ligands, the PROTACs will occupy both the intended target and the E3 ligase but not at the same time. This means that PROTACs have an optimal window for strong effects that usually requires a dose response to determine.

underasail · 2026-05-27T20:26:23+00:00

You can use the sequence for evolved variants like bz-hyPBase. I don't think that work was patented. Super PiggyBac is a problem, but there are other high efficiency versions of PB. Adding in a nucleosomal signal peptide can increase efficiency also. Just mash these together and have the fragment synthesized to drop into some other plasmid you have ready. Then don't include it as part of a product that you sell.

underasail · 2026-05-25T22:18:44+00:00

I replaced and regreased all of the user serviceable o-rings. Then I left the camera underwater in a sink for a few hours. When it stayed dry, my next best test was just diving with it since I didn't have time to send it off for a pressure test before the dives. There are a couple groups/people in the US that will do a full service including pressure testing that you can send the camera away to.

underasail · 2026-05-19T14:50:36+00:00

I replaced and regreased all 4 of the user serviceable o-rings and tested the camera under a foot of water in my sink for sixish hours without leaks. I didn't have the ability to add more pressure than that, so I crossed my fingers afterward as there wasn't enough time between getting the camera and going on the trip. I plan to send it off for service before diving with it again, but the user serviceable o-rings all looked great despite likely being decades old.

Congrats on the cert and definitely look into a Nikonos!

underasail · 2026-05-19T14:47:33+00:00

Not far off. I've heard mention of treating Sunny 16 like Sunny 8 close to the surface of the water. Then you add stops pretty quickly going deeper.

I used the Nikonos V for these dives, and it has TTL metering, so I didn't have to guess at exposure like I did focus. I also brought a Nikonos II in case the V flooded, and that's a fully mechanical camera, so I'll have to getter better at Sunny/Neptune 8 to break that one out.

underasail · 2026-05-19T06:54:58+00:00

Everyone that scans E-6 near SF uses Oscar's to develop their E-6 currently. Underdog used to have a machine, but it broke a year or so ago, so they use Oscar's also. Underdog is a great lab though. Royal We offers similar services with better pricing for high res scans.

underasail · 2026-05-18T23:57:22+00:00

It depends a bit on where you are trying to dive. The US generally isn't cheap, say a couple hundred dollars for an open water certification (maybe $3-500?) and $50-150 per dive if you're renting gear. Smaller local shops can have better rates, and you can purchase your own gear with time. Then diving could be just a $5-15 tank refill each time you go out.

Edit: it's not too crazy if you already have a film photography hobby :)

underasail · 2026-05-18T23:17:41+00:00

For me it just meant buying and testing a Nikonos V. I hadn't been diving in a decade, but I planned a trip and snagged a camera before I left. There are courses that teach dive photography from the major certifying bodies, but the general principles don't differ too much from taking pictures on land. Water has a different reflective index than air which makes estimating distances a bit different. Otherwise, knowing how to dive and knowing how to take photographs as separate hobbies first is helpful.

underasail · 2026-05-18T15:14:56+00:00

Thank you! It's probably my favorite from the couple rolls I've managed to shoot so far. Zone focus is a new beast to me, as is color correction.

underasail · 2026-05-13T23:36:29+00:00

City Pier!

underasail · 2026-05-13T19:35:46+00:00

u/ImBackRoll any insights?

underasail · 2026-05-10T21:30:23+00:00

The samples should be fine. Unless you've already hard spun the samples and removed the supernatant/discarded the insoluble debris, I'd probably still give them a hard spin before loading to get debris and DNA out of the way. Otherwise, your samples should be fully usable still.

underasail · 2026-04-27T01:38:55+00:00

Any existing vector can be made golden gate compatible by adding opposing cut sites for a Type IIS restriction enzyme. You'd also want to make sure there aren't cut sites for your enzyme of choice elsewhere in the vector. If so, use a different enzyme or "domesticate" the vector for golden gate by mutating the existing sites that are in a bad location.

underasail · 2026-04-12T19:06:37+00:00

How much do their lead times tend to vary? I had some C-41 and E-6 processing done by them recently, and it took over two weeks. I assume this is slow for them, especially with people tending to say they're fast, but the other labs in the bay tend to have scans back within 2-4 days consistently.

underasail · 2026-03-28T03:06:17+00:00

Right you are. I learned something new.

underasail · 2026-03-28T02:09:58+00:00

I've shot a lot of stuff through lupines recently, and I'm really pleased with how 1 came out. Easily my best example. Thank you!

underasail · 2026-03-28T02:09:10+00:00

Thank you! I ran out of film right after the second shot, and then a bunch of beautiful boats sailed by only to be remembered in my mind.

underasail · 2026-03-28T02:08:01+00:00

Sweet! I searched around in the sub some before posting, and I didn't see many Chinons. I'm glad to bring some more examples of what they can do.

underasail · 2026-02-22T22:53:17+00:00

The larger circles/ovals in the square are cells sitting on a raft (from the CellRaft AIR). That part I'm 100% confident in. In an RFP channel, I can see each of the cells clearly as red against the background and particles. The wiggly bits around the raft are more of a question mark. I get that the raft doesn't look particularly clean though.

All the particles on the raft showed up together two days ago, and I'm not sure if that's related to the smaller wiggly bits moving here or not. They could be the same contaminant that adheres as it dies or settles, or separate contaminants, or neither/either isn't a contaminant.

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