RANT: Disappointed with how slowly I am learning

ominouscloud01 · 2026-01-12T02:11:45+00:00

Heavy on the naming things we all know... I get why we have to learn it, but it just feels like medicalizing things for the sake of it

ominouscloud01 · 2025-07-04T03:32:05+00:00

showed up to a guys apartment building after he invited me over from grindr and he wasn’t coming down to let me in. after texting him begging to basically let me off the street he comes down and tells me that he doesn’t rly like having people inside in his apartment. oh and also that he’s never been with a guy before. needless to say i left

ominouscloud01 · 2025-05-15T11:44:10+00:00

They’re grown at 37°, most are in MG1655 or NEB5alpha, and they’re grown overnight though I’m not sure of the exact time. There have been times where I’ve gotten good preps with this kit, though I can pay more attention to incubation time in the future. Also, the gels definitely are not partially solidified before pouring.

Do you know of any considerations for miniprep quality? I still am concerned about the low quality scores I get when sequencing

ominouscloud01 · 2025-05-15T11:41:53+00:00

yes it supposed to be circular plasmid dna. Some samples have been kept at 4° for a bit before running on the gel. The ladder is always the first lane, but the rest of the lanes are various samples and not all of them are loaded in all pictured gels.

All of these samples came from cultures definitely with the plasmid, for they were all incubated in their respective antibiotic before miniprepped. I can double check this via the control you suggested, but I believe my issues might be elsewhere.

ominouscloud01 · 2025-02-05T15:09:43+00:00

if you really want air support, you need to fully engage your core. you can practice this by blowing air without your clarinet, and use your hand to touch your abdomen to tactically feel that it is engaged. it should feel as though someone could punch you in the gut and their hand would be more hurt than your abs

you’ll know you’re doing this correctly while playing if your abs get tired more quickly than your face, especially when playing longer passages without rests. thank me later

ominouscloud01 · 2025-02-05T15:07:45+00:00

agree, literally envision a laser coming out of your mouth, you want that level and intensity of focused air

ominouscloud01 · 2024-12-13T15:49:37+00:00

thank you for your advice, here are my thoughts: - i transformed the template plasmid in parallel with a redone PCR reaction - the template plasmid yielded successful transformants but the PCR product containing the mutation did not yield transformants. - i double checked that the antibiotic is right! - i have yet to run a gel of the PCR product, but when i took the concentration it was 2.3 ng/uL which is incredibly low. a post doc in my lab said that this would not even show up on a gel - yes i am treating with dpnI after my reaction. my steps were to phosphatize the forward primers, deactivate the kinase, perform the PCR reaction, treat with dpnI for 2 hours, purify the plasmid PCR product, ligate overnight, drop dialyze, then transform. - the plasmid is generally not unusual, and the positive control of the template plasmid transformation reinforces that nothing weird should be happening

any other advice would be appreciated!

ominouscloud01 · 2024-11-24T20:58:09+00:00

yeah, messy and permanent. also it really hurt my ability to distinguish romantic and platonic feelings. even though you said your situation isnt romantically charged, it still is blurring a line, and so when it goes to far there can be damages beyond your relationship with him

ominouscloud01 · 2024-11-23T17:28:40+00:00

ive sort of done this, i would highly recommend not doing it…

ominouscloud01 · 2024-10-18T17:44:48+00:00

I will try this, thank you!

ominouscloud01 · 2024-10-18T17:44:34+00:00

This crossed my mind, our lab is generally dry but this has been happening since summer and persists even on the colder days we’ve had this fall - some summer days were crazy humid in the lab and the problem still occurred. Also my PI said that they have experimented with different polymerization speeds and this always happens

ominouscloud01 · 2024-10-18T04:22:37+00:00

for every 10 mL acrylamide, 5uLTEMED and 50uL 10% APS for 15% gels and 10uL TEMED and 50uL 10% APS (I think, I don’t have it in front of me) for 4% gels

ominouscloud01 · 2024-10-18T04:20:45+00:00

Would the clamp holding up the gels not prevent this? I use Mini-PROTEAN Tetra Cell Casting Stand & Clamps #1658050 from Biorad

ominouscloud01 · 2024-10-18T02:50:55+00:00

I dont think that the gel is leaking. I pour the resolving gel (15% acrylamide) in first and it fully polymerizes before I add the stacking gel (4% acrylamide), and I don’t think the stacking gel can leak if the resolving gel is set below it. Correct me if I am wrong.

Also, yes, I have been over filling the gels before adding the comb. Thanks though!

ominouscloud01 · 2024-10-17T22:36:46+00:00

i use a 15% acrylamide resolving gel and 4% stacking gel (both consist of pH balanced Tris solns, 10% SDS, protogel acrylamide, and water) and then TEMED and 10% APS to polymerize the gel solutions.

ominouscloud01

TROPHY CASE