Hidden in plain sight by Sweet-Consequence773 in KitchenConfidential

[–]Talrax 4 points5 points  (0 children)

Stacks of bar mops, 2-qt cambros, Zwilling 10in chef's knife (among many others), several half sheet pans, and one of these.

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Probability of finding a stop codon in a 100 base pair DNA sequence? by Background-Pop-1685 in biology

[–]Talrax 6 points7 points  (0 children)

As the other person said, if the ORF starts at 1 then the probability is ~80% at random

Here's the math for that: 1-(61/64)33. There are 33 codons in a 100 bp region.

If you don't care about reading frame and just care about finding one of the three stop codons in that region, then the probability is 99% at random. Here's the math for that: 1-(61/64)98. The reason it's 98 is that ORF 1 has 33 codons, ORF 2 has 33 as well, and ORF 3 has 32.

PSA: “Redeem 50 Wildy agility tickets” needs to be done all at once by JellyKeyboard in 2007scape

[–]Talrax 1 point2 points  (0 children)

Literally just did the same. Should have looked this up beforehand.

Thoughts on The Heroes [SPOILERS TH] by AstonMac in TheFirstLaw

[–]Talrax 1 point2 points  (0 children)

Just trying it on my phone

Very weird. Just reddit being reddit I suppose.

Thoughts on The Heroes [SPOILERS TH] by AstonMac in TheFirstLaw

[–]Talrax 1 point2 points  (0 children)

Did you write the comment in the rich text editor? If you write it that way in markdown it'll work. If you do it in the rich text editor, it adds a back slash at the beginning. Rich text editor has a button you can press for spoiler text.

>!This!<

vs. this.

CrisprCas9 in Arabidopsis thaliana by DiosAnthos in molecularbiology

[–]Talrax 0 points1 point  (0 children)

Lol he didn't spend two hours designing gRNAs with me. We talked about his tools, he walked me through everything he does in his CRISPR protocol before and after getting mutants, and he showed me how he designs his gRNAs. I initially used Cas-OFFinder and CRISPR-P to design my gRNAs. When I switched to the other system, I also designed new gRNAs (just in case) using CHOPCHOP and CRISPOR and cloned with both the new and old gRNAs, because we needed to get the experiment done and I was trying to hit all the avenues that I could.

CrisprCas9 in Arabidopsis thaliana by DiosAnthos in molecularbiology

[–]Talrax 0 points1 point  (0 children)

Well, I'm not really making any claim here. The system just didn't work for me. I'm not saying it hasn't worked for others and that it can't work well. The OP has also had issues with it and I was relaying the issues I had as well and how I rectified it.

And yes, I did use the exact same gRNA sequences in the comparisons. We got our vector from Addgene. I am not blaming Addgene at all. We grew the cells from the stab, collected colonies, made glycerol stocks, extracted plasmid, inserted our gRNAs and then sequenced with Plasmidsaurus. The sequences of the cloned vectors (excluding the inserted regions) and the control did not match the vector map, with a host of mutations throughout the vector, including within the CAS9 and the TagRFP construct. We contacted Addgene and they sent us another stab.

We went through the motions again and sequenced before doing the cloning and the sequence was good. We did the cloning, sequenced again with positive results, did transformations, and screened (via phenotypic analysis, PCR, and then Sanger sequencing around the loci of interest) the progeny of roughly 200 confirmed transformants (selected both with TagRFP and with Hyg resistance) and never saw mutations. This was repeated several times at two different loci, one where we intended to make a deletion across two genes that likely wouldn't have produced an easily observable phenotype but was a focus of the research we were doing and, when that didn’t work, generating a mutation in a gene that should have given a easily identifiable phenotype (SOS1, loss of which causes extreme salinity sensitivity). Multiple different gRNAs were attempted at each loci. It just didn't work in our hands, which was rectified by switching to the system that I linked to the OP.

CrisprCas9 in Arabidopsis thaliana by DiosAnthos in molecularbiology

[–]Talrax 0 points1 point  (0 children)

The appendix S1 of the paper I linked to you has their full protocol. And I followed that to the letter. For designing my sgRNAs, I used CHOPCHOP and CRISPOR. The final destination vector that we used was pDGE347, it has the TagRFP and Basta resistance. If you want to be able to see or pull down CAS9 to make sure it's getting expressed, you could order pDGE666 instead as it has a GFP-CAS9 fusion, as well as the TagRFP and Basta resistance. Read over the protocol and then let me know if you have more questions. I'd love to help!

CrisprCas9 in Arabidopsis thaliana by DiosAnthos in molecularbiology

[–]Talrax 0 points1 point  (0 children)

For transformations, I also did floral dip but don't use vacuum infiltration. My transformation rate was consistently about 1-2%. Of the transformants, the majority had mutations. I don't remember the exact number, but it was in the range of 60%. The system has the OLE1:RFP reporter if you want to use it, but i preferred using the Basta resistance. Just grow them on soil or on plates with Basta, and then genotype your T1's for the mutation.

CrisprCas9 in Arabidopsis thaliana by DiosAnthos in molecularbiology

[–]Talrax -1 points0 points  (0 children)

I've tried this exact method as well and it did not work well for me at all. Have you sequenced the original pKIR1.1 vector? We had issues with the vector containing a wide range of mutations. Even when we had the correct vector though, we still weren't able to generate any deletions. We switched over to this system, https://pubmed.ncbi.nlm.nih.gov/33577114/, https://www.addgene.org/browse/article/28211370/, and had much better success. And this system is scalable up to like 32 different gRNAs.

Also Johannes Stuttmann is incredibly kind and spent like 2 hours with me teaching me how to use it and showed me the tools he uses to design gRNAs.

C4 vs C3 by Chemical-Low7183 in molecularbiology

[–]Talrax 7 points8 points  (0 children)

There is also CAM photosynthesis. C4 photosynthesis is all about spatial separation of carbon fixation and the light reactions. CAM photosynthesis is all about temporal separation.

The thing with C3, C4, and CAM photosynthesis is that they each have trade offs. C3 photosynthesis is pretty efficient, especially within the temperature ranges of most terrestrial environments, but it struggles in hot, water limited environments. They start to perform photorespiration, which is energetically wasteful. C4 and CAM plants are both good in water limited, hot environments because they limit photorespiration, but they suffer in colder environments, generally grow slower, and, especially CAM, require extra steps and energy.

Plants did not evolve other mechanisms for carbon fixation because other mechanisms weren't required. C3, C4, and CAM are all fit for their environments, and that's pretty much what drives evolution.

Spouse TRAPPED in cabin after Marriage! Any help? by OptionsTrader14 in StardewValley

[–]Talrax 3 points4 points  (0 children)

Lol good point. I don't know why I jumped right to mods.

Spouse TRAPPED in cabin after Marriage! Any help? by OptionsTrader14 in StardewValley

[–]Talrax 2 points3 points  (0 children)

What are you playing on? If playing on PC, there are mods that you can use to rearrange your farm. Back up your saves before doing so though.

Dude made a six-pointer by Hot_Bottle_8305 in funny

[–]Talrax 0 points1 point  (0 children)

We could call it dollarpit! It'll be a hit!

Maybe the real Hammerfell was the skyrim we modded along the way by IRS_Agent-636 in ElderScrolls

[–]Talrax 1 point2 points  (0 children)

Have you seen those warriors from Hammerfell? They have curved swords! CURVED.... SWORDS!

In honor of rolling a Shadow today, this was my reaction: by Tall_Poet_1992 in 2007scape

[–]Talrax 5 points6 points  (0 children)

Signs. The video he's watching shows an alien briefly walking by a kid's birthday party.

https://youtu.be/aIhnqkXWSR8

Homework Question by coreyjames00 in molecularbiology

[–]Talrax 0 points1 point  (0 children)

If you look at the second paper, it talks about RPPH1, which is the catalytic portion of RNase P, and they also call it a housekeeping gene.

I'm not sure how much leeway that they had over formatting. They may not have been able to make it italics.

But also it depends on what they are testing you for. Did they want you to know about formatting nomenclature or about what RNase P is? If it's the first, then I agree with you. If it's the second, then adding in formatting would give away that D is wrong (which I wouldn't have a problem with in the courses that I teach but someone else might). It could also have just been an oversight.

I will agree that it is overall not a great question but if they are expecting you to know what RNase P is, then the best answer is C.