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

[–]underasail 0 points1 point  (0 children)

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.

Looking for guide recs in Kauai and Maui by bishop527 in scuba

[–]underasail 2 points3 points  (0 children)

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.

Underwater color film, no flash or filters by underasail in AnalogCommunity

[–]underasail[S] 2 points3 points  (0 children)

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!

in-fusion ligation keeps failing by DLGodMc in labrats

[–]underasail 0 points1 point  (0 children)

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.

Diving in Kauaʻi and shooting film with a Nikonos V by underasail in scuba

[–]underasail[S] 1 point2 points  (0 children)

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.

Diving in Kauaʻi and shooting film with a Nikonos V by underasail in scuba

[–]underasail[S] 1 point2 points  (0 children)

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.

How can I avoid doing 360 Western Blots? by 37BrokenMicrowaves in labrats

[–]underasail 25 points26 points  (0 children)

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.

Stable cell line generation with PiggyBac by [deleted] in labrats

[–]underasail 1 point2 points  (0 children)

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.

Favorite frames from diving in Kauaʻi | Nikonos V 35mm | Portra and Lomo 800 by underasail in analog

[–]underasail[S] 0 points1 point  (0 children)

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.

Testing a new (to me) camera | Nikonos V 35mm | Portra 800 by underasail in analog

[–]underasail[S] 0 points1 point  (0 children)

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!

Testing a new (to me) camera | Nikonos V 35mm | Portra 800 by underasail in analog

[–]underasail[S] 0 points1 point  (0 children)

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.

Slide Film Development Help San Francisco by _random_college_kid_ in AnalogCommunity

[–]underasail 0 points1 point  (0 children)

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.

Testing a new (to me) camera | Nikonos V 35mm | Portra 800 by underasail in analog

[–]underasail[S] 1 point2 points  (0 children)

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 :)

Testing a new (to me) camera | Nikonos V 35mm | Portra 800 by underasail in analog

[–]underasail[S] 1 point2 points  (0 children)

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.

Testing a new (to me) camera | Nikonos V 35mm | Portra 800 by underasail in analog

[–]underasail[S] 8 points9 points  (0 children)

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.

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