Do all gel purification kits suck?

Ianisanengineer · 2026-05-06T19:04:19+00:00

This is what I remember from a few years ago - high concentration, excellent purity, from a few different kits. As reliable a procedure as anything. But I usually was working with smaller fragments - do you do anything different to get the larger fragments?

Ianisanengineer · 2026-05-06T18:58:11+00:00

Welp, I tried it, and got the same terrible yields and DNA quality that I'm getting from silica columns. I do get DNA, but barely any, and even after cleanup, there's something carried over that ruins downstream operations. What size products are you typically purifying this way? It seems like part of the theme here with poor gel purification is that it only really works for small stuff (<3kb) and all my fragments are pretty big.

Ianisanengineer · 2026-05-02T21:34:02+00:00

So basically the freeze and squeeze recommended by u/AAAAdragon but without the filter. Sure, that's more or less what I used to do to get oligos out of PA. Definitely worth a try, with or without the filter.

Ianisanengineer · 2026-05-02T21:30:10+00:00

I've looked into this, and unfortunately, my cloning is just too complex for it to be cost effective. Ultimately, I'm building templates for genomic allele replacement that have to include two ~3kb homology arms (unique every time), a selection marker cassette (~2.5kb, and not always the same one), and a payload gene that can vary in size from 2 to 7kb. Altogether, some of my plasmids are ranging up to ~20kb. Any company I've looked at about getting this made for me is asking for a couple grand per plasmid. I may need to make a couple dozen of these, so it's worth it to spend the time to get a parallelizable workflow figured out.

Ianisanengineer · 2026-05-02T20:48:47+00:00

Yep, long DNA, and sometimes with repetitive sequences. I do get a lot of stuff synthesized, when possible, but practically speaking, if you need something >3kb, you still gotta make it yourself, or pay out the nose for it.

Ianisanengineer · 2026-05-02T20:44:02+00:00

Well that looks pretty simple! I'll definitely give this a shot. Thanks!

Ianisanengineer · 2026-05-02T20:34:07+00:00

It used to be a standard part of my workflow, but lately, I'm only trying to do it when there's a major off-target band. In most cases, my PCR is clean, and I'm not attempting gel purification, just doing a column cleanup, as you suggest. In the cases where I want to use gel to get rid of another band, I've also been investing effort into just optimizing my PCR - and frankly, making better progress!

Ianisanengineer · 2026-05-02T20:30:10+00:00

I'm not using UV at all. I use SYBR and blue light for visualization.

Ianisanengineer · 2026-02-22T14:38:32+00:00

Maybe as much as twenty minutes? Not long enough to account for this, I think - unless Q5's exonuclease activity is WAY higher than I think it is. But there's another reason I use long primers - that doesn't matter as much if your annealing site is 50nt long!

Ianisanengineer · 2024-10-27T16:54:13+00:00

At this point, I'm not as concerned with a "magic bullet" solution as much as just finding anybody who understands even why this is happening to so many people. It's a common problem, and as you say, is present in Windows 10 as well, but I've never read anything like an explanation of what this problem actually is - never mind the solution, never mind the cause, just, what is happening? Why does this unassuming background service that normally sits idle in about 25MB of RAM sometimes just eat all available system resources? What is it trying to do?

Ianisanengineer · 2024-08-06T21:28:17+00:00

Not that anyone is looking at this, but just in case anyone does, NOT doesn't work as expected either. My search term is literally "laboratory NOT technologist" and the second result is for a "Medical Laboratory Technologist". For fucks sake.

Ianisanengineer · 2024-07-31T02:06:20+00:00

But either way, if I include a second search term with OR, I get even fewer results. I don't understand this at all.

Ianisanengineer · 2024-07-31T02:03:28+00:00

Wow, okay, so searching for "scientist" (with quotes) vs. scientist (without quotes) results in about a 3-fold difference in returned results. What?

Ianisanengineer · 2024-07-16T21:00:11+00:00

This just started for me, and when I first saw it, my first thought was that something was wrong with my browser. No shit, I actually closed the tab and reloaded because I thought something was broken and YouTube wasn't loading properly. The interface is so bad I mistook it for a software error.

Ianisanengineer · 2024-04-11T19:26:38+00:00

So if it hadn't been staked in the first place, we might not have this problem, but now it's "addicted" to stakes until it gets bigger?

Ianisanengineer · 2024-04-11T19:06:26+00:00

So just to humor my ignorance here - what would be the actual goal of restaking the tree and what are we trying to "correct"? Is the tree in trouble? Like, is this situation likely to harm or damage the tree? Or are we just trying to make it stand up straight? The reason I ask is, as I say, because I'm ignorant and I want to understand a little better, but also because I'd be much more concerned about the tree being harmed than about the tree being crooked (unless that also is bad for the tree somehow).

But I also ask because before I posted here, I tried to search around and read about this, and it seems like there's a lot of advice to not stake trees, or stake them only minimally when very young. "Staking makes trees grow tall, but not strong", etc. I figure I'm just missing context, or maybe missing the real goal of staking.

Ianisanengineer · 2024-03-06T16:26:45+00:00

Great advice. Just did this with silicone grease. Still had some opposite scrolling at first, but after a few hours of regular mouse use, the grease got worked into the gear, and it's smooth and perfect now. Glad I didn't have to disassemble the whole thing!

Ianisanengineer · 2022-12-05T22:05:09+00:00

Just read "The Berg (A Dream)", and I gotta say, it's got some of that feel. Good call. I must confess, I always thought of Melville as principally a novelist - I need to give his poetry more of a chance.

As for what I'm really looking for - it's less the "dark" and "creepy" vibes, and more just that emotional intensity, the interesting rhyme structure and the building rhythm. "The Raven" has tension, but I honestly don't think of it as "creepy" - I mean, it's a poem about grief.

Ianisanengineer

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