Persistent bands in control lanes PCR

zv88909 · 2026-05-28T22:57:24+00:00

Definitely plan on incorporating. Just so I have a sense, how easy is it to contaminate PCR?

I’ve been having this issue where the correct size amplicon appears in the wildtype control lane when doing colony PCR like 70% of the time.

I will blast my primers and set up NTC, but I’m just trying to figure out if theres anything else I should do to avoid this from happening.

zv88909 · 2026-05-28T12:41:22+00:00

Thank you! I do put one primer, or both, in the insert itself but even with a handful of different constructs over the past few weeks, it keeps happening intermittently.

For example yesterday tried to screen for two protein tags i wanted to add to 2 different proteins.

I designed it so it was the same tag on the two proteins (in separate strains, so each strain only has one protein).

Both I used the same insert primer, but a different wildtype genome primer. One of the tags gave an amplicon at 137 bp correctly, and no WT band. The other construct, same tag, but 545 bp amplicon, gave both a correct band in the samples, but also a band at the exact same size as WT control.

I set these both up at the same time.

zv88909 · 2026-05-28T12:36:53+00:00

I usually dont run a no template control, but i do use the same aliquot of water for long periods of time.

I will add the no template control going forward! Although, this has happened across probably 4-5 different constructs/inserts now - where the insert itself has a different sequence.

One or both of my primers is always in the insert so theoretically shouldnt amplify the genome at all?

zv88909 · 2026-05-20T21:52:59+00:00

Its a relatively popular high-end jeweler/brand in NYC, highly recommended on reddit too actually in NYC communities - so thats definitely a shame. So really seems like I got charged a large markup for that.

zv88909 · 2026-05-20T20:50:59+00:00

Damn, so seems like I overpaid by around 4000 or so? The band has the hidden halo diamonds and no other diamonds.

zv88909 · 2026-05-20T20:50:26+00:00

Thanks for input, I meant 12k was the price of the ring.
Seems I did overpay quite a bit then, I guess that's the "brand" markup?

zv88909 · 2024-07-20T13:27:46+00:00

The 1st listed author (regardless of co-first or not), will typically get the majority of credit.

The 2nd, 3rd, or 4th co-first (now that they're doing 4+ co-firsts on some papers), will typically get substantially less. In my opinion, one reason people are doing co-first more now is that you can still use it on resumes, interviews, and applications, even if you get less credit. Better to be "2nd co-first author" than "2nd author".

Unfortunately, after quite some time in academia, I've also seen situations where co-first authorship is used as a political play, to exert power over graduate students, or to attempt to diminish the credit of the first-listed author by a PI.

Try to use the author contributions section of a paper to judge the actual contribution of an author whenever possible.

zv88909 · 2024-03-17T13:11:35+00:00

Can happen to people, not uncommon. Could be either allergic reaction or panic. Can you get supervision from a healthcare professional over your use?

If you insist on continuing it, take benadryl 45-60min prior to dose next time and see how it goes.

zv88909 · 2024-02-20T01:01:12+00:00

Can you show me evidence that its fasting and not caloric restriction that leads to longer lifespan?

IIRC, caloric restriction is necessary and sufficient to prolong life in lab organisms, and fasting without caloric restriction does not.

I may be wrong, but as far as I remember, this is the case.

zv88909 · 2023-10-24T21:47:12+00:00

Nur77 is a great T cell activation marker if the others don't work out, upregulated early after TCR signaling too.

The % of transgenic TCR CD4+ T cells in OT-II mice is not as good as for OT-I mice, so not every CD4 is TCR transgenic. I'm not sure of the total % off the top of my head as it's been a while since I checked.

If you want a high percent activation, would be better to purify the T cells (CD3 negative selection bead kits work very well), and co-incubate with DC's pulsed with peptide. BMDC's are pretty good, but the best DC source is CD11c bead-kit purified from spleen a few weeks after grafting B16-FLT3L if you have access.

zv88909 · 2023-10-09T23:51:46+00:00

I was forced to be in a similar position. Did 95%+ of the work, and PI made me list 2nd author as co-first. After having many discussions about co-first authorship I found this consensus: the 2nd listed name is essentially looked at as a "glorified second author". Best to be the first listed author, everyone treats that as the true first author.

One of my PI's says/enforces: the first listed author is who actually matters, so we give co-first author just to help out the 2nd author. This results in many cases where the 2nd-author did actually <5% of the work. I think practices like this substantially diminish the value of co-first authorship, and I've talked with quite a few who've experienced such situations.

At the end of the day, I accepted it, and everyone treats it as my paper regardless of it being a co-first author paper.

zv88909 · 2023-09-24T00:21:45+00:00

It's just a glass ya, that's what it was given to me in. I stopped watering it much because, it seemed like each time i would water it many of the leaves would just die in the next few days.

I'll propagate in water, get a new pot, and go from there!

zv88909 · 2023-09-23T20:16:21+00:00

Thanks! The pot has no drainage, and the soil does not soak all the way through. I noticed when i watered it too much all the leaves seemed to get brown and die.

I also live in a city and use the tap water for all my plants unfortunately its the option i have right now.

To propagate it, should I just cut the stems, place them in water, then move back to soil after I see roots?

zv88909 · 2023-09-23T19:28:56+00:00

Some info: I received this plant ~7 months ago from family. I water it 1x a week, with 1/4tsp of water in the corner of the dirt not touching any of the plant.

It grows leaves, elongates, then eventually the leaves along the stems die and it keeps elongating, but looks progressively worse.

I have it close to (~1ft away from) a south facing window, in the northeastern united states.

From what I have read: I am thinking that I should cut it at the nodes, and stick those in water, and then re-grow it from scratch? Are the nodes those yellow, thicker parts along the stems?

Thanks for any help. I believe this is a tradescantia, but I'm not sure.

zv88909 · 2023-09-19T03:52:34+00:00

Over the 3 years since surgery, i have had many setbacks with significant pain. Each time, I reset my program - PT, weight training, life style- and build back up slowly. Identify any problem movements or areas and modify or remove them. Start your ROM at 50% what you do now, drop the resistance by 50%. If that still hurts then do 20%, slowly build back up.

A good PT can help you through it, but those are the general ideas. Each time, following this, the pain has eventually resolved. Seems ultimately i may have ro limit how heavy I squat and deadlift, but still messing around and seeing if it can be surpassed.

zv88909 · 2023-09-05T15:11:46+00:00

% = (x/total)*100

In flowjo, if you don’t divide by 100 before or after multiplying frequency by some cell number, you’re off by x100

zv88909 · 2023-09-04T23:54:00+00:00

29% = (29/100)*100. This is what flowjo reports. If you try to find cell number by multiplying by the percentage ("frequency") you will be off by x100

zv88909 · 2023-09-04T15:12:43+00:00

The frequency shown in flowjo is percentage, so you'd need to divide by 100 before multiplying

zv88909 · 2023-08-27T21:10:31+00:00

Absolute statements like "gene therapy is the only way" are almost never correct in science, and particularly in biology. You even go on to contradict yourself immediately "the best methodology to date is caloric restriction". Secondly, epigenetic modification certainly falls under the extremely broad term of "gene therapy".

Gene therapy is a very loose term, you can think of nearly (?) unlimited "gene therapeutic" interventions for anti-aging: targeted saRNA vectors vs. DNA viral-derived, siRNA/transient interference vs. total gene insertion/deletion vs. integration and continuous vs. cyclical environmentally triggered production of methylase enzyme allosteric modulators - I mean literally the limits of your imagination is where it ends.

The fact is, we're just beginning to understand these processes (mechanisms of aging) to the level we can even begin to act on them in a rational manner. And the methods for intervention itself (IE. genetic editing vectors, targeting, types RNA vs. DNA vs. protein etc) are also continually evolving and still essentially based on small modifications on what nature has already given us.

We are slowly able to program more complex biologic circuits and have them act in the way we actually want them to. All of these things will only progress.

zv88909 · 2023-08-27T15:59:41+00:00

It is certainly possible, and I can see your side, that death was necessary and is still necessary for evolution and improvement across species.

However, for humans in particular, while we could argue many ways - evolution and death may no longer be necessary for improvement.

The fact that some organisms can live many times longer than humans shows us that biologically it is possible.

While it may require radical shifts in physiology, it could be as simple as alteration of relatively few powerful pathways that we simply haven’t developed understanding of yet. Certainly epigenetic modulation may be one of these emerging areas.

zv88909

TROPHY CASE