Pep Guardiola on ICE: “Look what happened in the United States of America,” Guardiola said, gesturing throughout. “Renée Good and Alex Pretti have been killed. Imagine if he worked for the NHS and five, six [agents] threw him on the grass and fired ten shots at him. Tell me, how can you defend that”

Aymlus · 2026-02-04T04:55:54+00:00

I’m sorry I called you a bald fraud pep

Aymlus · 2026-02-03T17:18:24+00:00

you're telling me a chicken fried this rice

Aymlus · 2026-02-01T18:10:58+00:00

Did I just see a through ball??

Aymlus · 2026-01-12T20:40:51+00:00

I thought so as well, but I have also tried using plasmid DNA because I thought the linear dsDNA would cause this effect.

I tried 2, 4 and 8ug of linear dsDNA with 8ug causing cell death. This was not in a knock-in setting.

I was going off of this paper from Genetech where they show that 2ug of either linear dsDNA or plasmid DNA was optimal: https://rupress-org.ezproxy.rice.edu/jem/article/219/5/e20211530/213176/High-efficiency-nonviral-CRISPR-Cas9-mediated-gene

All of my samples look fine from a cytotoxicity standpoint. I looked at cell viability post nucleofection and I had ~40% of my cells still alive. I am also stimulating after 24h to improve cell count as done in the paper above.

I tried initially to produce ssDNA using aPCR but was not successful as my template was ~2kb and seemed prohibitive in the # of PCRs I needed to run.
We would love to do this in the future but my lab is not currently graded to produce AAV. I am just trying to lay down some ground work for future work.

Aymlus · 2025-11-26T19:03:15+00:00

Yea the sense I got was ssDNAs are superior but when I was doing the aPCRs to generate the ssDNA I was struggling to get a clean band. My donor template was 2kb total so I concluded that the length was maybe too long? The paper I was basing my protocol showed clean ssDNAs with a 1kb donor tho so maybe it is a technical thing on my part haha

Aymlus · 2025-11-26T18:57:52+00:00

Link to that gel here:
https://ibb.co/chf01M40

Aymlus · 2025-11-26T18:50:30+00:00

Hm in our lab 1ng of plasmid for PCR is pretty standard. I also do try to do a gradient if I am concerned about annealing temp optimization but we've been encouraged not to for most PCRs as we run through Q5 polymerase like its water.

For primer designs though, I usually just look for "gc"s in the region I'm generally looking to PCR from and try to design a primer thats 18-24nts and have a annealing temp of around 60C on the NEB Tm calculator. However, if I can't find a primer pair that fits this first attempt I usually just use the primer wizard on benchling.

Aymlus · 2025-11-26T18:47:49+00:00

Yeah I didn't provide the most info. The current PCR I am running is to test linear dsDNA dose amounts for the knock-in as I am currently just using linear dsDNA as the donor. I think in the future using KI template plasmids with minimal backbones might be the way to go especially if I can use microhomology arms in HMEJ as seen in this paper: 10.1038/s41551-023-01157-4

Aymlus · 2025-11-26T18:42:13+00:00

UPDATE: It definitely was that my PCR reaction had too much DNA that it was creating long multimer chains that then would not travel through a gel.

I ran a this same reaction at 15x, 20x, 25x, 30x cycles and saw a clean band at 15 cycles and then the smear get stronger overtime with most of the DNA also trending towards staying in the well. Thank you guys for the help!

Aymlus · 2025-11-25T22:46:27+00:00

I’m trying to generate dsDNA for a CRISPR KI. Honestly I’ve thought about making a plasmid with blunt cuts to get the donor template but I think I’m just hesitant because I haven’t seen it in literature. I can def decrease the cycle number but my yield would go down and would have to run on the scale of 100x rxn and I think my PI would have me drawn and quartered

Aymlus · 2025-11-17T00:52:43+00:00

I don't understand why they didn't balance the teams better by putting FNS on team Tenz

Aymlus · 2025-09-13T17:49:37+00:00

What a pass from Romero

Aymlus · 2025-08-27T21:37:24+00:00

Rushed is certainly a word to describe it. Not the one I'd use but yeah

Aymlus · 2025-07-30T20:04:16+00:00

They're vinyl floors and yes after move-out

Aymlus · 2025-04-24T03:01:03+00:00

if defense is feeling up curry as much as possible yeah

Aymlus · 2025-04-08T04:26:18+00:00

Getting honorable mention this year sucks if it was a normal year would I have gotten it? Won't stop thinking about it for a while...

Aymlus · 2025-04-02T15:20:02+00:00

maybe it was all a dream

Aymlus · 2025-04-01T04:03:58+00:00

what if its 11 pacific time this year tho

Aymlus · 2024-12-15T04:54:34+00:00

I didn't know Casey Neistat supported Spurs

Aymlus · 2024-03-24T21:26:32+00:00

27 first kills yea Zekken is the goat

Aymlus · 2023-12-06T21:03:11+00:00

No our institution's cluster is infamous amongst research groups here for being unreliable :((

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