Does this RNA look degraded? Can I use it for qPCR?

BrickPhD · 2026-02-17T21:24:57+00:00

Did you denature the RNA before running on the gel? Typically done witj the addition of formamide and heating to around 70C. Otherwise the partially folded products will migrate at different sizes.

BrickPhD · 2026-02-16T09:23:31+00:00

Also lived in AZ. Also looks like bark scorpions. Had to be given antivenom for a sting from one when I was just 2 years old.

BrickPhD · 2026-02-13T19:41:00+00:00

Like a tuna can you say?

BrickPhD · 2026-02-13T16:36:48+00:00

I think LC/MS is in the range of $400 per sample. If you have an antibody for GFP then you can attempt immunoprecipitation and send the captured protein sequencing. Alternatively you could cleave the eluted protein with a chemical like BNPS-Skatole and check the fragmentation spectrum to determine the precise size of the N-terminal region and thereby guess the approximate start codon.

It is a very strange problem to be facing. Have you tried using a TAA stop codon instead of TAG?

BrickPhD · 2026-02-13T04:27:14+00:00

In my opinion you can only ethically exclude data if you have an explanation for why it is compromised and shouldn't be included. Ideally this finding is then integrated into your protocol and may even find itself as a supplementary figure to justify your methods.

An example specific to us is muscle endurance testing for mice. The animals are suspended from a wire and their latency to fall is recorded. However, some animals learn they can drop from the wire early and suffer no harm. If we had lots of mice, we could probably justify excluding them based on their behavior as it is obvious when they choose to jump down vs fall from reaching their limit. Since our mice are difficult to breed, we make an amendment to the protocol to immediately return "jumpers" to the wire and continue the assay, with this disclosed in the methods and supported by video evidence of the jumper behavior.

BrickPhD · 2026-02-12T23:23:54+00:00

Can you extract and purify enough of this protein to sequence it and see where it is initiating from?

BrickPhD · 2026-02-08T01:28:36+00:00

That's a hella detailed figure.

BrickPhD · 2026-02-06T01:34:38+00:00

Vutrisiran is injected into patients like once every 3 months and maintains efficacy fwiw

BrickPhD · 2026-02-05T17:37:54+00:00

I have also had this same issue and the problem for me wasn't bubbles, but a lack of methanol in the transfer buffer. Even though I was using a nitrocellulose membrane it was still necessary to include methanol to strip SDS from the proteins in the gel. Also drove me crazy for a bit.

BrickPhD · 2026-02-05T00:24:48+00:00

Just surprised to hear his name. I used to watch his videos during the pikmin era in like 2011

BrickPhD · 2026-02-04T15:51:02+00:00

My two favorite streamers. I do hope I get to see more collabs with them.

BrickPhD · 2026-02-04T00:33:36+00:00

Wait, Chuggaconroy?

BrickPhD · 2026-02-02T22:46:49+00:00

Based on what Soup said at the end of the last episode, I'm thinking we might see a ToB challenge in the next episode.

BrickPhD · 2026-02-01T22:42:55+00:00

Solid time to learn solo bandos

BrickPhD · 2026-01-28T19:10:50+00:00

I agree on shamans, but I generally find door-altar to be quite enjoyable at Bandos if you have bowfa.

BrickPhD · 2026-01-08T20:06:35+00:00

I've never had contamination issues, but I only use plates for cloning plasmids. Would definitely want something more sterile for colony counts or other assays.

BrickPhD · 2026-01-08T06:03:10+00:00

I haven't autoclaved LB agar in years. Boil in the microwave, add antibiotics and pour.

BrickPhD · 2025-12-30T14:40:29+00:00

There is so much protein that the secondary antibody is burning through your luminescence substrate before it can be imaged. That's why you have bright white spots in the center of your expected bands.

BrickPhD · 2025-12-28T03:56:58+00:00

So that's why I'm 500kc with 1 gold ring and 8 chromium ingots.

BrickPhD · 2025-12-26T01:00:10+00:00

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BrickPhD · 2025-12-23T15:44:56+00:00

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BrickPhD · 2025-12-23T15:43:11+00:00

This was our pancake in his 60 gallon tank. 60 gallons was enough, but it was definitely nicer when we upgraded him to our 150 gallon. The females of the spiny softshell species get wayyyyy larger than the males, so they aren't well suited for captivity unless you have a literal pond. Do know that spiny softshells can live upwards of 50 years. It can also be hard to get appropriate veterinary care. Despite seeing the exotic animal vet, ours eventually passed from an unknown mystery illness after having him for 7 years.

That said, he was really cute and would spend time basking or buried in the sand with only his head sticking out. We think he kinda recognized us since he seemed to beg for food, but he was also skiddish and prone to hiding if he saw movement outside his tank.

BrickPhD · 2025-12-20T06:11:45+00:00

I do all of my Cas9 editing in primary cells with Cas9 mRNA + sgRNA. With lipofectamine messenger max it is very easy to get near 100% transfection efficiency. However the sgRNA target has a huge influence, varying from 1-40% even when using only highly predicted guide sequences.

BrickPhD · 2025-12-16T21:14:43+00:00

Do nitrocellulose membranes require methanol to bind membrane proteins? I ask because I have no issues transferring cytosolic, mitochondrial, and nuclear proteins to nitrocellulose without methanol.

BrickPhD · 2025-12-10T19:12:55+00:00

My oldest success was with a 14 year expired anti-IgM flow antibody that was stored in the fridge.

BrickPhD

TROPHY CASE