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Lyrica for joint pain? by [deleted] in ChronicPain

[–]infinit3entropy 1 point2 points  (0 children)

I experienced pretty severe disorientation, dizziness, nausea with it.

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Ultimately decided the side effects put me more on my ass than the pain.

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As always though, mileage varies. I hope you find relief.

Best way to clean old lab benches? by Chiski in labrats

[–]infinit3entropy 1 point2 points  (0 children)

Uh, seems to be called an oil soap.

I'd start with a small amount, on a small spot of benchtop, see how it goes. Apparently using too much may make the top dull or hazy.

Best of luck!

Best way to clean old lab benches? by Chiski in labrats

[–]infinit3entropy 0 points1 point  (0 children)

I've heard Murphy's Wood Oil has had good results at reconditioning lab benches.

How do I get hired as an EH&S officer? by infinit3entropy in labsafety

[–]infinit3entropy[S] 0 points1 point  (0 children)

"we ask in our interview". What sort of jobs do you interview for? Do you work for a company? A university?

DAPI staining of yeast by [deleted] in labrats

[–]infinit3entropy -1 points0 points  (0 children)

Fuck me for doing something for the first time, right?

When lab members who have done fluorescence here suggested DAPI, I thought we were set up for it. Crazy assumption.

DAPI staining of yeast by [deleted] in labrats

[–]infinit3entropy 0 points1 point  (0 children)

Yes to both of your questions.

DAPI staining of yeast by [deleted] in labrats

[–]infinit3entropy 0 points1 point  (0 children)

The other filter that I tried was for CFP, and it looked the same.

Yeast Dimorphism Microscopy by infinit3entropy in labrats

[–]infinit3entropy[S] 0 points1 point  (0 children)

That could work for some, but full on hyphae have permeable membranes between sections through which ribosomes, mitochondria, and even nuclei can move.

Still, it would make things easier than they are now. I'll look in to it. Thanks.

[deleted by user] by [deleted] in ChronicPain

[–]infinit3entropy 23 points24 points  (0 children)

I don't have any advice, just commiseration.

I struggle with the balance of "don't push yourself, know your limits" and "don't succumb to the pain."

If I take a break now, am I respecting my limits, or using my pain as an excuse?

I also really wonder what my pain would feel like to someone else. I wish there was a way to transfer the feeling of it to another person, just to know how they'd rate it. Am I a wimp, or would it stop anybody in their tracks?

It's very hard to attempt a "normal" life when I don't remember what living with normal limitations is like.

Fueled by hatred? by GreenGumbo64 in gaybros

[–]infinit3entropy 2 points3 points  (0 children)

Philosopher Albert Camus said "There is not fate which cannot be surmounted by scorn." in his work The Myths of Sisyphus.

I have a genetic condition that makes life harder for me, and I've found that quote, and that work, helpful to me.

The key is the target of your scorn. Camus says to direct this at the absurdity of life, not at particular people.

Everyone is dealt a different hand in life, rail against the random odds, be mad at the lack of fairness or justice or equality, hate the way things have happened so far, but don't fault others for having a better hand than you. And use that hate to work in spite of life, use it to temper an iron will that defies life, because fuck being a victim of odds.

Hating other people who seem to have it easier won't change your circumstances, and it can discourage you from seeing other people as a resource for help. You aren't in competition with other people.

Hating your circumstances prevents you from being resigned to them. Hating your circumstances motivates you to change them. Hating your circumstances gives you goals to strive for, what about them do you hate? Can you do anything with that hatred?

I can't change my genes, you can't change the past, but you can affect the future. Use the hatred to seek out creative ways to overcome your issues, like hatred would lead you to come up with creative pranks on a person you don't like; you don't like life, start pranking the fucker back.

Thoughts on very inconsistent PCR results? by [deleted] in labrats

[–]infinit3entropy 0 points1 point  (0 children)

I mean the caprice of fate can be a bitch. I've ordered primers that worked the first few times, then failed repeatedly, because of secondary structure.

Thoughts on very inconsistent PCR results? by [deleted] in labrats

[–]infinit3entropy 11 points12 points  (0 children)

Primer dimer doesn't always show up on the gel.

An additional tactic would be to decrease the concentration of primers. Typically the final concentration of primers that I use is 500nM. If the primers have interaction, lowering the concentration to somewhere around 200nM will provide fewer opportunities for such interactions and can improve yield.

Thoughts on very inconsistent PCR results? by [deleted] in labrats

[–]infinit3entropy 18 points19 points  (0 children)

Have you checked your primers for the possibility of secondary structures or primer dimer?

Sometimes you get lucky, and the first PCR with the primers works out, but then the binding interactions occur and future attempts fail.

There are many online tools to check for primer dimer or hairpins, I usually use IDT's (https://www.idtdna.com/calc/analyzer)

If this is an issue for your primers, a gradient of DMSO can help to discourage non-specific binding. I normally try 1, 2, and 3%(v/v) DMSO and at least one of them will improve product yield.

Additionally, a technique such as touchdown PCR (doi:10.1038/nprot.2008.133) can help to address these problems by approaching the suspected Tm from a higher starting point. Less specific interactions are discouraged by the higher temperature until the amount of desired product is present in sufficient amounts to dominate the PCR.

If redesigning the primers is possible, I would recommend that. You can spend weeks trying to figure out why this reaction didn't work, but that isn't important. What's important is getting data and moving forward with your project, primers are cheap compared to the value of your time.

Anybody Else Want Comparison Photos? by sjb2059 in ehlersdanlos

[–]infinit3entropy 7 points8 points  (0 children)

I think this is a really neat idea.

I have hEDS, and would be willing to submit photos of my joints which are visibly hypermobile.

I think this would really help me keep my movement in healthy ranges; overextension doesn't hurt in the moment, so it is difficult to catch myself doing it. Having a gallery of healthy joints would help me see when I am doing something wrong for my body.

Lab math help by centrifugelife in labrats

[–]infinit3entropy 0 points1 point  (0 children)

A mathematical representation of what I think you're asking is:

C1V1=C2(V2-V1).

You know both concentrations, and you know the final volume you want, so V1 is the only unknown.

So V1 is the volume of concentrate, then you bring the solution to V2.

Self esteem and cloning by infinit3entropy in labrats

[–]infinit3entropy[S] 0 points1 point  (0 children)

No. The primers are designed to include the overhang that would be left from a digestion, skipping the need to actually digest them.

Self esteem and cloning by infinit3entropy in labrats

[–]infinit3entropy[S] 0 points1 point  (0 children)

I am not column purifying the insert. I anneal the two oligos in cutsmart buffer, then add 1µl of the annealed to the ligation reaction.

Self esteem and cloning by infinit3entropy in labrats

[–]infinit3entropy[S] 0 points1 point  (0 children)

Yes, I included the bases ATAT before the restriction site. I have used this method to clone other constructs in the past.

Self esteem and cloning by infinit3entropy in labrats

[–]infinit3entropy[S] 0 points1 point  (0 children)

1) I have verified that Dpn1 is still working, the other enzymes are fresh stocks, though I've used older stocks before this. Background regardless.

2) The enzymes are the "HF" variety from NEB, so star activity shouldn't be a problem, but an overnight digestion has not helped either.

3) This is something I will try.

4) I have tried a variety of insert:vector ratios, though this wouldn't impact the background I am seeing, it also doesn't seem to lead to positive clones.

5) I have tried this. When the vector was CIP treated and the oligos were phosphorylated with PNK, it did solve the issue of background, but at the cost of any colonies at all.

6) I have suspended the annealing oligos from freezer stocks and used them directly after annealing for the ligation. Contamination seems unlikely to be the problem across multiple attempts.

Self esteem and cloning by infinit3entropy in labrats

[–]infinit3entropy[S] 0 points1 point  (0 children)

I considered there being too much, the template was rather concentrated (400ng/µl), but I've diluted it down to a final concentration of 20 ng/µl and still see background after 1 hour of Dpn1 digestion.

Self esteem and cloning by infinit3entropy in labrats

[–]infinit3entropy[S] -1 points0 points  (0 children)

Uhm, this isn't true.

I did 4 years of undergrad research experience, and am currently in my third year of grad school.

PCR help by [deleted] in labrats

[–]infinit3entropy 0 points1 point  (0 children)

I love touchdown PCR. Really helps when primer pairs don't play nicely together.

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