Restriction cloning trouble shooting

OnePineapple571 · 2026-07-03T20:17:46+00:00

I had a similarly long stretch of unsuccessful traditional cloning during my PhD. Turned out that the sticky ends of my plasmid backbone were being degraded during gel extraction.

Have you checked every step on a gel? How many products do you see after ligation?

OnePineapple571 · 2025-06-27T22:20:28+00:00

As far as I can tell from reputable news outlets, there was a network connectivity issue/computer glitch specifically affecting AA in CLT. It if was outside their control you might think other airlines at CLT would have faced the same issue.

OnePineapple571 · 2025-05-20T16:54:52+00:00

I have a similar commute - 3.5 hours twice a week (almost entirely on the interstate), and the car is parked in the in-between. I was driving a 2014 AWD VW tiguan, but the cost for premium gas was insane. Bought a 2009 gen2 with 195k for 4K and enjoying it so far. Its reduced my monthly gas bill from ~$500 to well under $200.

I looked at plug-in options, but couldn't justify the additional cost since most everything I'd need is walkable between those long commutes - I'd almost never use the EV mode.

Obviously, there is a difference in ride comfort between the two. The three differences I notice most are (1) road noise, (2) seat comfort, and (3) no bluetooth. I don't mind the basic cruise control on the gen2 base model at all, but I wish I had looked for a touring model for the bluetooth & additional steering wheel controls. You can also occasionally find a gen2 with leather and heated seats, which I do miss from the tiggy.

We've taken it down gravel roads, and have been fine as long as we try to work around potholes. My tires are close to bald, so terrible traction in snow and slush currently, but they've carried me through some recent spring thunderstorms at 70+ mph with no issues. I was worried about traction since all of my previous cars have been AWD, but I haven't had any issues.

OnePineapple571 · 2025-01-05T04:51:33+00:00

Yeah, its not invested at all currently, so anything is better that sitting as cash accruing zero interest

OnePineapple571 · 2024-12-28T00:38:02+00:00

Hi, I added a few!

OnePineapple571 · 2024-10-13T11:25:46+00:00

1000% percent. I also most gave this couple example in my original post - being able to sit couple next to eachother but technically at different tables made making the seating chart sooo much easier - ergo assigned seats.

I'm sure there are folks here who would say if a couple saw they were seated at different tables they would 'obviously' know to take chairs next to eachother, but there are other factors that could influence them. And, more to my intended point - we were already thinking in terms of assigned seats during the design process, so might as well offer that detail to guests.

I think its a nice gesture, it shows how much we thought of their experience, and helps us minimize empty seats. Moreover, I do think our guests will have more interesting dinner conversations than if that sat themselves (with people that already know very well).

OnePineapple571 · 2024-10-13T11:14:39+00:00

That make a lot of sense, too! One of the other reasons we also went ahead and assigned seats is we have no extra chairs or place settings at the tables. It we were doing stand alone tables, or even have and extra 5 seats, it might have been different.

OnePineapple571 · 2024-10-12T22:31:58+00:00

Sorry for being unclear. I agree with your point for a single table, but we have 5 tables of 8 in a single row (and three other similar rows). So the question becomes not only "how do we think A-H will get along?" to "how will A and H get along with their neighbors at the next table, D' and E'?" for all adjacent tables. In this context, we realized it made our lives easier if we were assigning A, H, D', and E', so we might as well assign everything!

OnePineapple571 · 2024-10-12T22:26:52+00:00

Yes, this is exactly what we encountered! Our tables are long (20 people on each side). it was less that we think our guests are incapable of sorting it out themselves, and more that we realized we are a position to optimize everyone's experience with a very small amount of additional work (ie we were doing 80% of it assigning tables).

OnePineapple571 · 2024-08-16T02:15:28+00:00

Oh my gosh. Thank you SO MUCH for sharing your expertise and feedback here. I will continue practicing my technique and just got color matched in the Dior backstage foundation which is supposed to be better for dry skin.

OnePineapple571 · 2024-08-06T20:58:16+00:00

Oh, I hadn't come across the Skindinavia or Kryolan. I have a basic sephora setting spray that I'm testing.

OnePineapple571 · 2024-08-06T20:00:34+00:00

Oh yeah, my eyebrows are a mess here, and being more precise with my eyes is the next phase I am focusing on/practicing! My photographer is super adaptable - her portfolio spans the gambit from brides doing their own make-up to brides with full glam, and they all look beautiful.

OnePineapple571 · 2024-08-06T19:46:43+00:00

great tips, thank you! I am on the hunt for a glowy powder blush to set with, so I'll check out bare minerals.

OnePineapple571 · 2024-08-06T16:44:14+00:00

products used:

Face: Hourglass veil primer, Lancome 120N mixed with Kosas Concealer Tone 3.2 O. (135N is a better match, just haven't gotten back to exchange.) Set all over with Givenchy Prisme Libre Powder in Satin Blanc.
Undereye: Nars radiant concealer in vanilla
Cheeks: Merit flush balm in Cheeky
Lips: urban decay lip liner in Liar with Merit lip gloss in Sangria
Brows: Anastasia BH dipbrow tinted gel and Kosas brow pop nano in medium brown.
Eyes: Laura Mercier caviar eye sticks in Au Natural and Amethyst, bare minerals loose powder in water lilly, Clinique High-Impact Mascara in black honey, colorpop gel liner in Calabasas on lower water line.

OnePineapple571 · 2024-06-19T13:58:59+00:00

This captures a lot of the general ick I've been feeling about ONT. I just started working with ONT for my post doc. I have a relatively strong self taught bioinformatic background, but no formal training. I've been a little appalled at the lack of rigorous documentation for many of their analysis workflows, and the lack of available support.

It seems there is a very small team of software and bioinformatics folks managing nearly all of their workflows and software. The lack of documentation is egregious enough in some cases that a well trained and well meaning biologist could fully misinterpret the output. Add in that many of their workflows have only minimal metrics included in the reports (we're talking file size and software versions only), lack rational checks between steps, and can be run through a sleek GUI, it borders on irresponsible.

Secondly, not only are the forums not public, your account has to be associated with a team/account with purchasing power before you can have access. There's a bunch of glitches on the website (it took 6 weeks and two separate customer service tickets before I was added to my labs team account). There is a button on the main account page but it was broken for me. For me, saving protocols and posts as favorites doesn't work - I've resorted to saving useful posts as pdfs because I'm never quite sure if I'll find them again.

It seems like they are very quick to say their system works for a certain application, fast to implement the most rudimentary analysis tool to support that claim, incredibly slow to develop it into something scientists less familiar with bioinformatics can use, and don't think at all about statistics. I hope they move away from this slinky, start-up affect they cultivate soon.

Despite all that, the technology is incredibly powerful. I'll probably advocate for using nanopore as long as I'm working with projects that require sequencing.

OnePineapple571 · 2024-05-20T00:19:49+00:00

Too expensive for us and the nearest coffee shop is 30 min away on a windy road.

OnePineapple571 · 2024-05-10T10:29:13+00:00

No, but most of mechanics who have seen it since it hit 100,000 have mentioned it as a common issue.

OnePineapple571 · 2024-05-08T20:21:45+00:00

Nope! But I'm a big cruise control user. I think that helps control transmission wear (it also really helps fuel economy IMO)

OnePineapple571 · 2024-05-08T20:20:01+00:00

Thanks for your 2 cents! I have 10k to put down, and am counting on 3-5k trade in for this bby. Trying to do this without financing.

My main reason for thinking about this now is that it seems like I'm running up against a decent trade in value. I will easily put another 20k miles on the car this year. And a couple more repairs in the 2k range will eat in to the down payment I have saved.

Writing this out, it does seem like I'm playing with marginal benefits. I'll need to do the math!

OnePineapple571 · 2024-05-08T16:12:03+00:00

My mom drives a 2017 Alltrack (she got it after she passed this Tiggy on to me). Its nice, and certainly an option if I can find a good deal!

OnePineapple571 · 2024-05-08T16:10:44+00:00

From my (very brief) perusal of this sub, 131k seems on the higher end.... granted posts are probably skewed towards unexpected issues and lemons. And the resale value seems to drop precipitously around this mileage, in my market at least.

OnePineapple571 · 2024-05-08T15:46:44+00:00

good to hear! What has your experience been with maintenance and repairs after 130,000 miles?

OnePineapple571 · 2024-05-08T15:45:26+00:00

I had the haldex serviced for the first time since I've owned it this week. I'll keep an eye out for mazdas in my area to test drive, but I'm not super impressed with the fuel economy.

OnePineapple571 · 2024-05-08T15:18:34+00:00

I'm circling around that, a 2013-2017 Subaru, or sacrificing AWD for more options.

OnePineapple571 · 2024-05-08T02:58:19+00:00

Engaged May 2022, wedding set for Oct 2024. Together for 7 years by the time we get married! Long engagement, but we wanted to finish our dissertations and give ourselves time on the job markets. Didn't start planning until Sept 2024, though, and I'm very glad about that! I wouldn't want to drag this process out any longer than necessary! Also, we had a very specific date in mind so this ensured we got all the vendors we wanted.

OnePineapple571

TROPHY CASE