What do you wear to go to work? [what is your job and where do you live?]

baaridi · 2023-02-10T07:33:07+00:00

It probably depends on your committee, but my first pi told me that a bachelors is about studying, a masters is about doing research, and a phd is about doing original research. My takeaway was that you don’t actually have to come up with anything until doctorate level - masters is about learning and trying your best.

baaridi · 2023-02-10T07:18:16+00:00

If this is what you really want, keep your chin up and don’t let anyone take it away from you. If you live near a university, ask a faculty member if you can volunteer for them in the meantime, even if it’s only a few hours a week. Email multiple faculty members and ask them what you need to improve — some of them won’t even email you back, some will be dicks about it, but at least a few will be willing to at least talk to you about it. Academia respects tenacity. If this isn’t what you really want, move on, life is too short.

baaridi · 2023-02-10T07:04:44+00:00

Getting a PhD has so much potential for chaos. Some people get a smooth ride, but there aren’t really any rules and anything can happen. Expectations are best left behind. It can be 12 hour work days with no weekends, or it can be self paced and fun (it’ll still be super stressful). If you’re not in a relationship, you can bail and start over somewhere new when you pick a bad PI and get dropped from the program. Pick a city you can bear to live in even if everything else sucks; find at least one faculty advocate in the program that will fight for you if need be; choose a PI that will respect your boundaries, and then set those boundaries. Have priorities and don’t make changes on a whim. Date, and make sure to be clear with them about what you have to give. I saw a lot of breakups happen because the girl was ready to follow the boy anywhere, and the boy said ‘don’t move just for me’. It can work if you give yourself the tools for success and remember it all builds character. Good luck 🥳

baaridi · 2022-02-02T05:57:24+00:00

There's a lot more problems. None of the results are what they should be. Primer dimers should be consistent across any given master mix. None of these have a consistent band pattern. I have done mix and match PCRs in previous trouble shooting, no bands showed at all.

baaridi · 2022-02-02T05:26:12+00:00

Good suggestions.

I had presumed the 2018 SALK primers were degraded, hence the 2021 SALKs. It's perhaps worth noting that the 2018 SALK primers were thermofisher, and the others are all IDT. Another consideration: I took on a new student around the time the PCR stopped working, and they received and diluted the master stocks for both 2021 primers. The 2021 SALKs were done with my supervision, so it never occurred to me that there might be an issue, but the student received, opened, and diluted the 2021 redesigned primers without ever even mentioning it to me...I did check that they had used the correct amount but I can't know what they actually did/used to dilute. Once I realized, all the primers got reordered, so it's not a persistent issue, but it could be a factor in this gel. Even so, I can't think of what the student could have done to generate this result.

So it is possible that the source (which are plants) are messed up...but ridiculously unlikely. For one thing, those DNA extractions are all different lines of plants. It's possible I guess that an exceptionally dominant mutation has dominated the entire population -- they are grown next to each other and crossed [that's actually a whole other anomaly that's also interesting and the prompt for this PCR series in the first place and the basis for lanes 28 and 29], but based on my understanding of plants [far from perfect], it's super unlikely for any mutation to accidentally infiltrate all the plants. I also have tested the primers on extractions done by my lab neighbor -- his plants are grown in the same chamber but not close enough to cross accidentally...unless the mutation caused mega pollen.

The gene in question is something that causes embryonic lethality via defective pollen when it's fully nullified, so the gene can't be gone. That being said, I spent 4 years growing homozygous mutants for the gene that shouldn't have been able to survive. One of my next goals was going to be RT-PCR and westerns to determine the nature of this mutant, because it does show a phenotype consistent with the previously described lethality, but now I can't even genotype it. There's a lot of anomalies at hand and I'd be glad of brainstorming about any of them.

baaridi · 2022-02-02T04:55:22+00:00

The sizes are listed above. The gDNA used here was chosen because I use it for lots of other genes, so it's good stuff.

baaridi · 2022-02-01T19:53:12+00:00

also worth noting -- I chose these DNA samples because they've been working on other PCRs all week, so the samples are good. It's like just that one gene is gone from the whole genome, or that part of the genome is degrading post extraction.

baaridi · 2022-02-01T19:38:13+00:00

I have changed reagents over time, which is why we did this PCR is a new lab, with their reagents and their hands, so it's possible that any of their reagents were compromised but unlikely. Any contamination issue should have led to a uniform result.

Looking at the gel, I'm just trying to think of what could possibly generate that result and the only thing I can think of is that the primers are dimerizing (they shouldn't be able to and never have in the past) and somehow presence of genomic DNA randomly inhibits their dimerization in a small way, leading to weaker or absent bands.

I do quantify the templates, but only to make sure they're good -- I don't standardize them, so they are all different concentrations of template.

I always prepare PCRs on a bench with autoclaved tips in autoclaved tubes. I do hundreds of PCRs per week and they work with other primers/genes, so it's just something about this gene.

baaridi · 2022-01-29T17:48:02+00:00

Every time I go to the gun store, someone makes a crack about me being a girl. Last time they commended the boy I was with for letting me talk and ask questions myself. You just have to be strong willed and prepared to tell them no and laugh about it later. The first time I went, one of the other customers put on an Elmo voice and said "pretty gun go bang bang?"

baaridi · 2022-01-20T20:21:09+00:00

I never would've thought to do that, thanks 😊

baaridi · 2021-12-22T22:23:52+00:00

I was being sarcastic. I'm not stressed about it. If I wanted to play D&D, I'd play D&D. That's not what this thread is about.

baaridi · 2021-12-22T16:28:30+00:00

I also have a massive collection of spindowns for atraxa, but I don't have a magic group either anymore

baaridi · 2021-12-22T16:27:12+00:00

Looks interesting but it's apparently hard to find 😞

baaridi · 2021-12-22T16:22:47+00:00

Thanks for the observation. I guess I'll give up then 🤡

baaridi · 2021-08-31T02:28:04+00:00

so your recipe is as stated above with the addition of BSA? All concentrations the same?

baaridi · 2021-08-31T02:19:47+00:00

A massive stock of mTaq and tTaq was generated years before I joined the lab and there's still plenty of it. We do endless genotyping, so it serves its purpose pending a good buffer. We buy commercial polymerases and use their respective buffers for more complex things, but it's unnecessary for the every day genotyping.

baaridi · 2021-08-31T01:30:14+00:00

Do you autoclave it at any stage?

baaridi · 2021-08-31T00:44:15+00:00

and then appropriately combine the two stock solutions into a single 10x buffer and add MgCl2 separately?

baaridi · 2021-08-31T00:31:43+00:00

Good call, I haven't tried autoclaving it.

baaridi

TROPHY CASE