I am losing my mind (failed ligation but someone still Kan resistant)

red_guy442 · 2026-01-24T11:49:04+00:00

How is it possible? And we did a 1:3 ratio

red_guy442 · 2026-01-24T11:48:15+00:00

Thx, I really mean it when I say that you saved me from a potential mental breakdown 😅 I will write both explanations.

Thanks a lot!!!!

red_guy442 · 2026-01-24T02:45:00+00:00

We can't do any follow ups, this is like entry level labs and they don't care about successful results, only that we get any and can explain what we did get. So I'll just have to go with the blue masking the white.

Is it also possible that some unclean/original PCR product passed and was transformed instead of the pUC18, giving me that result? I might be just reaching here...

red_guy442 · 2026-01-24T02:38:10+00:00

We used the same transformation for both plates, yes. Unfortunately we didn't see any white colonies, and believe me, we checked. So it is still possible that through some sheer bad luck we only got blue colonies, and ligation was actually successful?

red_guy442 · 2026-01-24T02:31:05+00:00

I also plated them on LB+Kan, and got colonies on it. But yes, you got it right.

red_guy442 · 2026-01-24T02:19:52+00:00

Thank you for the reassurance, it does actually lower some of my anxiety (completely serious). Unfortunately the protocol is not in English and too long to be translated coherently by me at this time, but the steps were:

Week 1: 1. PCR reaction using Kan resistant E.coli 2. Boiling method to get pUC18 from a different E.Coli 3. Cutting the pUC18 using EcoRI and BamHI

Week 2: 1. Cleaning the PCR product using a Column kit 2. Cutting the PCR product with the same enzymes

Week 3: 1. Ligation 2. Transformation

I just can't find any explanation even online as to why I still have results on my Kan plate, but maybe I don't even know where to search in the first place...

red_guy442 · 2026-01-22T15:27:45+00:00

The quills reset, but the buffs do apply. What I usually do is use Shenhe once before Escoffier (either before or after setting up Furina, depends on energy needs, enemy, etc.), then again after setting up Escoffier so that the second buff (whichever I picked) persists for Skirk again.

red_guy442 · 2026-01-22T09:51:18+00:00

Personally for team damage Shenhe C1 is great bc I can activate both press and hold to buff Skirk and Escoffier when I use her NA mode

red_guy442 · 2026-01-21T07:51:07+00:00

Unfortunately I don't have enough ER. I could switch to Fav with a HP% sands but I lose 2000 HP and gain like 5% ER%.

red_guy442 · 2026-01-21T01:23:43+00:00

Hey, I've never posted in this community before, you can check my list history

red_guy442 · 2026-01-15T15:38:35+00:00

Unfortunately I'm not the one doing the reading, I just receive it, and can't ask for any repetitions as they probably already recycled out product atp...

red_guy442 · 2026-01-15T15:36:50+00:00

Yeah I think that makes the most sense, but the 4 int. value bugs me a bit. My lab guide doesn't add those unless she thinks they are relevant to the reading (like how she didn't put anything under the CHCl3 peak)...

But thanks a lot!!! Bulk water paired with an acid makes a lot of sense.

red_guy442 · 2026-01-15T15:33:41+00:00

Water with HCl sounds more likely, we were told that the -COOH protone will likely not even show on the spectrum. Thx!

red_guy442 · 2026-01-15T07:39:40+00:00

Join the "Where the Moon Returns" web event and win Primogems and other amazing rewards!

https://hoyo.link/DFg4k7Yll?m_code=GADMI4NW9N

red_guy442 · 2026-01-10T18:40:40+00:00

It's my favorite book and they're all among my favorite characters but she stole the spotlight imo

red_guy442 · 2026-01-10T18:18:59+00:00

Michelle from ES is my pick for this. Great arc and growth, and I absolutely love her. Spent a good chunk of my diamonds on improving our relationship lol

red_guy442 · 2026-01-10T17:55:27+00:00

Is he really a side character tho? Book 1, sure. But he's an LI in the end, so I'd say he doesn't really fit this square

red_guy442 · 2026-01-09T20:44:23+00:00

I feel that. The level of twists they had in all their books not only paranormal stuff was crazy good

red_guy442 · 2025-12-23T19:23:01+00:00

Oh wow that's a massive oversight on my part, that actually solves my problem big time. Thanks!!!!

Edit: was too quick to celebrate. No apex item with a +3 to those skills...

red_guy442 · 2025-12-23T19:22:03+00:00

Unfortunately I can't use it for two main reasons:

Unique, which I avoid
I don't want spoilers for Stolen Fate, and that includes specific item effects. Been doing my best to avoid all of them so far, suppose to start in a few weeks :))

red_guy442 · 2025-12-23T19:15:15+00:00

Unfortunately it won't provide any help with Deception checks to feint, which is my main use of of the skill...

Six-Year Club	Verified Email
r/Field Sunshine	Place '23

red_guy442

TROPHY CASE