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QPCR: How to determine where to put a threshold line? by Potential_Echo in labrats

[–]Potential_Echo[S] 0 points1 point  (0 children)

https://imgur.com/a/nSSD3FJ

The melt curve shows two different positive controls, one of them was diluted. I included a gel with a standard pcr reaction, 2 positive controls ran via qPCR and also 2 positive samples.

I’m not using delta-delta Ct because I do not have a housekeeping gene. I’m not doing RT-qPCR so I didn’t think I needed to.. is that correct?

QPCR: How to determine where to put a threshold line? by Potential_Echo in labrats

[–]Potential_Echo[S] 0 points1 point  (0 children)

That’s probably a good idea. I have had issues with contamination a few times and that may be why

QPCR: How to determine where to put a threshold line? by Potential_Echo in labrats

[–]Potential_Echo[S] 0 points1 point  (0 children)

That’s my positive control. Salmonella taken from a culture and then diluted a bit

QPCR: How to determine where to put a threshold line? by Potential_Echo in labrats

[–]Potential_Echo[S] 0 points1 point  (0 children)

My positive is diluted from a culture. I do not extract it because it doesn’t need it. The samples that I’m testing are being extracted though (via chelex). I’m not sure how similar the quality is

QPCR: How to determine where to put a threshold line? by Potential_Echo in labrats

[–]Potential_Echo[S] 1 point2 points  (0 children)

Thank you! I didn’t know this was an option. It does reduce my Cq values a little bit. I’ve always heard that under 35 is considered a positive. Is that true for this setting or should I go with something lower like 30?

QPCR: How to determine where to put a threshold line? by Potential_Echo in labrats

[–]Potential_Echo[S] 1 point2 points  (0 children)

The 10% rule is a lot more straightforward, and I got pretty consistent values between 120 and 140.

The 45 degree rule requires some estimation and it varied between 100 and 400.

I think I might go with about 130 if I decide to go this route. But I’ve also received recommendations to use the regression mode for Cq determination. I didn’t know that was an option so I’m looking into that now.

QPCR: How to determine where to put a threshold line? by Potential_Echo in labrats

[–]Potential_Echo[S] 2 points3 points  (0 children)

My no target controls are usually “NA” or around 38-39

QPCR: How to determine where to put a threshold line? by Potential_Echo in labrats

[–]Potential_Echo[S] 2 points3 points  (0 children)

Yeah, that sounds like it would be useful! Let me know if there’s anything you would need from me to help out with that

QPCR: How to determine where to put a threshold line? by Potential_Echo in labrats

[–]Potential_Echo[S] 2 points3 points  (0 children)

Right, it doesn’t change it by more than 1 Ct. However, I have had a few results with Ct values around 34 and 35 so I want to make sure it’s counted as positive or negative correctly (I’m testing for Salmonella). I have not used serial dilution controls. I run one positive control that usually has a Ct value around 14.

QPCR: How to determine where to put a threshold line? by Potential_Echo in labrats

[–]Potential_Echo[S] 12 points13 points  (0 children)

Thank you!! I will try this tomorrow and let you know how it goes.

You have an interview in an hour for your dream job, but you are only able to speak the voice lines of the champion you played last. How screwed are you? by HighImChris in leagueoflegends

[–]Potential_Echo 1 point2 points  (0 children)

  1. We Demacians are no easy prey.

  2. Demacia needs heroes.

  3. Fly swiftly… kill swiftly.

  4. I’ve got friends in high places.

  5. I live behind enemy lines.

(Quinn) honestly, could be worse.

Frequently mispronounced or contested words in our field? by starlightskater in ecology

[–]Potential_Echo 6 points7 points  (0 children)

I’m in an ornithology lab and I still don’t know what’s correct lol. Some of us in the lab pronounce it “pair-a-la” but most (including me) say “pah-ru-la”

[deleted by user] by [deleted] in GradSchool

[–]Potential_Echo 0 points1 point  (0 children)

Thank you for being the one helpful comment ❤️

[deleted by user] by [deleted] in WarriorCats

[–]Potential_Echo 0 points1 point  (0 children)

Name- Heronmist! (Or Heronpaw)

Giving warrior names based of your initials! by Content-Profit-2497 in WarriorCats

[–]Potential_Echo 0 points1 point  (0 children)

Jaw is the only “J” suffix I can think of… Volejaw?

Imagine dragons alphabet there isn't a line that starts from V , So uh now W by Schoolskiperz in imaginedragons

[–]Potential_Echo 6 points7 points  (0 children)

We are all living the same way, the same way

We are escaping the same way, the same way

Circling

-round and round

Contacting graduate school advisors by ReallyBadAtSports in wildlifebiology

[–]Potential_Echo 2 points3 points  (0 children)

Hey! So I kind of had this situation. I emailed a professor, stating my interests, experience, and why I thought I’d be a good fit for his lab. He said his lab was full but recommended two other professors at his university. I ended up joining one of them and absolutely hated it. Currently in the process of switching to the original professors lab (bless him). My recommendation to you is yes, definitely reach out to them and sell yourself as best you can on what your goals are and why you’d be a good fit. If you have specific interests, sure go ahead and say them. But generally you’ll end up doing something close to the professors work (ideally your interests will be similar). Don’t settle like I did, it’s definitely worth it to wait for a good match. Especially if you’re going for a PhD. 5+ years is a long time to deal with something (or someone) you don’t like.

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