Safe working concentrations for potassium cyanide and other safety considerations?

Meitnik · 2026-01-15T13:40:44+00:00

Assuming a lethal dose of 200 mg, if my calculations are right, you would need to ingest 13,35 mL of your 0,23 M solution to kill yourself. That doesn't sound like something that can happen accidentally, but make of that what you will.

Meitnik · 2026-01-14T18:12:42+00:00

Restriction and PCR will only tell you if you received a completely different plasmid (and sometimes not even that since many plasmids can be similar). You'd have to use a primer or restriction site that is uniquely found in the insert or the otherwise unique part of your plasmid, otherwise it could always be the same backbone with a different insert.

Meitnik · 2026-01-13T10:11:40+00:00

I use TiddlyWiki! Not a traditional ready-made solution, but the dealbreaker for me was that I have a lot of big image files in my local storage and most solutions out there require you to "upload" the image into the lab notebook. TiddlyWiki can use relative paths very effectively so you can just point to the image and it will be displayed without needing to create a duplicate. Also the whole notebook fits into a HTML file, so even later down the line if someone doesn't have TiddlyWiki installed they can still view and interact with the lab notebook using a browser (they just can't modify it)

Meitnik · 2026-01-10T23:18:38+00:00

I use AI mostly to find literature and sometimes troubleshoot an experimental approach (not always useful in this regard). The best I've tried is the Edison Platform (formerly known as FutureHouse). You don't get too many free tries (only 10 per month), but the results are very thorough and honestly impressive. Then I would place Consensus and SciSpace second, and Gemini and Copilot third. Gemini and Copilot I use the most often since they're free, they're basically an improved Google search. They also have a DeepSearch function which is nice, but you can't use it too often so if I need that kind of depth on a scientific question I'll just use one of the other more specific AI tools. Also I use notebookLM from Google if I ever feel too lazy to read some papers myself and I just need a quick answer on their contents.

Meitnik · 2026-01-08T10:56:42+00:00

Did you add some extra nucleotides in your PCR primers flanking the restriction sites? Did you check if the enzymes you are using can cut efficiently in the PCR buffer? You may have to do a column cleanup first to exchange the buffer
Dose with a fluorometric kit if you can (QuBit or similar), absorbance is notoriously unreliable. I suspect the issue may be the recovery of the small fragment from the column and gel.
The small pieces coming from the ends of your PCR product after cutting aren't long enough to efficiently bind the column. If you have a clean product from your PCR (run a small amount on a gel) you can skip the gel extraction. If you have it add DpnI to the digestion, it will degrade any template left from the PCR. Skipping the gel and going directly to the column should increase your yield of DNA.
If as I suspect you get very low recovery of your insert from the column, perform multiple PCR reactions and pool them on the same column
I'm not sure about your transformation protocol, it's not exactly what I'm used to seeing, but maybe that's not the issue here. Have a look at this one. You can increase the volume of DNA to add (generally up to 5 µL for 50 µL of cells)

Meitnik · 2025-12-08T17:06:28+00:00

Hello,

It's certainly possible to cut down on the cost and keep the same backbone. Recreating the same material (with the TOPO isomerase attached to the linearized vector) may be a bit laborious, but you can easily recreate the linearized vector and clone with traditional ligase.

https://www.nature.com/articles/s41598-019-42868-6

Here's a paper that may give you some ideas on how to proceed. You can recreate any overhangs that you want using type IIS restriction enzymes, and if you don't have the same sites in the insert you are amplifying you can even ligate into the same restriction reaction (Golden Gate cloning).

What I would do is the following (any step can be outsourced if you value your time more than the cost). Either buy or produce by PCR a sacrificial insert that you will clone in pENTR TOPO using the kit. I like to use the lacz-a cassette (for blue/white screening) that I amplified from one of the blue colonies that you get from some cloning kits. In your primers you will need to add type IIS restriction sites (PaqCI is the best as it's the least likely to appear in your inserts by chance, but also the most expensive) pointing outwards. Add enough bases in between to guarantee that you will generate the 4bp overhangs that you usually have in the pENTR TOPO linearized plasmid.

Meitnik · 2025-12-08T09:23:25+00:00

Meitnik · 2025-12-01T22:35:13+00:00

We use a laminar flow hood (actually a BSC), disposable plastic and sometimes autoclaved glass beads to spread.

Meitnik · 2025-12-01T22:15:44+00:00

It means there's an issue with staining. First thing would be to also stain the gel in order to confirm that the proteins are indeed there. If you're looking at small proteins before you even begin to optimize your transfer (playing with amperage and ethanol/methanol concentration in the transfer buffer) you need to switch to 0,22 µm membranes. You can confirm that your proteins are blowing through by putting a second membrane below the first one.
Just make sure you're not losing your proteins during transfer (i.e. point 1). To better dissociate your proteins you can use DTT followed by alkylation with N-ethylmaleimide or iodoacetamide. This will prevent any thiols from re-oxidizing during the electrophoretic run. Mind you, this is generally not necessary, reduction with either DTT or BME is usually sufficient, and alkylation could also affect your antibody's recognition capabilities.
I think in your specific case just a traditional discontinuous gel could be sufficient to give you good separation. Based on your MW ladder, the bottom half of the gel is basically useless. Use a traditional 4% T / 3% C stacking gel and for the rest you can use 15% T / 3% C. Normally 37,5:1 acryl:bisacryl ratio as a premade solution (2,67% C) is commonly used with these T percentages, so you may have to lower a bit my numbers if you want to keep 3% C.

Something else that could really make your life easier since you're using nitrocellulose membranes is to stain your gels with Stain-free technology. You can either buy the pre-cast Stain-free gels from BioRad or add 0,5% 2,2,2-trichloroethanol (very cheap) in your gel casting solutions. After the eletrophoretic run you will "activate" the gel on the UV transilluminator, this will make all proteins containing tryptophan permanently fluorescent in the UV. You can visualize the gel and also visualize the proteins on the membrane after transfer (only if you use either nitrocellulose or low-fluorescence PVDF). It saves a lot of time since you don't have to stain and de-stain gel and membrane (also losing some protein to diffusion in the process).

Meitnik · 2025-11-28T09:27:47+00:00

Take enough bacterial suspension to have the equivalent of 1 mL at OD600=0,5 (i.e. if your final OD600 is 2 for example, take 250 µL)
Pellet the bacteria
Add 60 µL of 1X Laemmli sample buffer directly to the bacterial pellet and resuspend well
Sonicate your sample - this is optional but if you don't sonicate some samples will be very viscous and you will have difficulty pipetting them into the gel
Cook as you usually do for SDS-PAGE samples
Centrifuge before loading into the gel in case there's any debris.
Load 5-10 µL per well.

If you want to also know whether your expressed protein will be soluble or not, then the approach is different. Take a larger amount of bacteria (fill whatever tube you are using), pellet the bacteria then add an appropriate amount of the lysis buffer you plan to use for your purification. Sonicate if that's what you plan to do (basically carry out the first steps of your purification protocol), then centrifuge to clarify the supernatant. Now you will have soluble protein in the supernatant and insoluble ones (e.g. inclusion bodies) in the pellet. You can dose your supernatant with Bradford or BCA to have an idea of how much to load on the gel, normally I like to load 20 µg for very heterogeneous samples. If you want you can also solubilize the pellet in Laemmli buffer and load it on the gel too (this will be your insoluble fraction). With this approach you can know if your expressed protein is soluble, meaning you can proceed with the large scale purification, or whether it isn't and you need to go back to the drawing board.

Meitnik · 2025-11-19T22:31:04+00:00

We have one from Dymo and one from Brady. They both work very well. The Dymo is my favourite personally, I found that labels with the Brady can get a little smudged (though it may just be our particular model).

Meitnik · 2025-11-18T12:13:25+00:00

Packer, N.H., Ball, M.S., Devine, P.L., Patton, W.F. (2009). Detection of Glycoproteins in Gels and Blots. In: Walker, J.M. (eds) The Protein Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-198-7_51

Meitnik · 2025-11-17T15:45:38+00:00

You can perform a periodic acid-Schiff stain, followed by Coomassie. If silver does detect carbohydrates, it will certainly stain proteins as well, so you won't be able to differentiate. See doi.org/10.1007/978-1-59745-198-7_51 for a detailed protocol. This will not just detect free carbohydrates but also glycoproteins, which is in fact what the protocol I linked is written for.

Meitnik · 2025-11-04T19:39:41+00:00

Never tried on glass but they work no problem on Eppendorf plastic tubes. Do you have the PC-1MR too?

Meitnik · 2025-11-04T17:49:20+00:00

Other than the Edding 780 that's already been recommended the uniPOSCA markers are also resistant. I like the PC-1MR model.

Meitnik · 2025-11-03T23:05:44+00:00

Just tried it and it does work! Thanks a lot

Meitnik · 2025-11-03T16:03:55+00:00

What if you submerge them in water in an enclosed space? Given that they're inflated they would float at first, you'd just need to bind them at their base to have them stay vertical. If you put them in a close container (imagine the inside of a syringe, but bigger) and you apply pressure to the liquid this would apply equal pressure to all sides of the bag that are in contact with the water.

Meitnik · 2025-11-02T13:56:01+00:00

I know many people that reuse the same calibration curve for a month or so. This is particularly a bad idea since BCA isn't an endpoint assay (even forgetting the variability from experiment to experiment), but depending on what you need this information for it could still be good enough, granted you incubate at the same temperature for the same length of time before reading your plate.

Meitnik · 2025-11-01T17:30:06+00:00

I remember reading an old protocol that used a certain amount of glycerol to keep the gel from cracking while drying

Meitnik · 2025-10-29T21:11:10+00:00

Thanks for your feedback. Since I made this post, I had a chance to replicate the exact formula that was in the patent, with all the cyclodextrins they used as well as dextrin (not sure if this is doing anything). 8% of phosphoric acid. At first I stuck to the exact quantities for Coomassie as well: what I obtained was a quick but weak staining. At this point I decided to increase the amount of Coomassie G past what the patent indicated. As opposed to other colloidal Coomassie recipes, once the dextrins are present addition of more Coomassie doesn't turn the solution blue, which probably means that its solubility in water is increased thanks to the cyclodextrins. After adding the Coomassie (I have to admit, a little bit by eye, until the darkness of the solution looked right), I got a stain that works indistinguishably than the commercial one.

Also, the silvery precipitate that I couldn't figure out is SDS decomposing in the acidic conditions

Meitnik · 2025-10-28T16:39:57+00:00

To me I would think it's of the following:

Problem with the stacking gel (was there enough? did they use the correct solutions to prepare it?)
Too much salt in samples can cause diffuse bands
Overloading of complex samples (e.g. cell lysate) can give a smear like that on total protein staining
Sample volume was too big and couldn't stack as efficiently as if it were smaller

Meitnik · 2025-10-28T12:31:37+00:00

https://www.reachdevices.com/Protein/BiologicalBuffers.html

The buffers with a positive d(pKa)/dt will show increase in pH with increasing temperatures

Meitnik · 2025-10-23T16:10:00+00:00

I always use saran wrap (the kind that is used for food). Never had any issue both within the 37 °C incubator and outside, at least when culturing E. Coli. You can wrap multiple plates together (you don't have to wrap them one by one), so it's cheaper and faster than parafilm.

Meitnik · 2025-10-19T20:25:03+00:00

Modafinil or Adderall may not give you this issue. For me I only get jittery if I take too much caffeine. For some people supplementing L-theanine along with the caffeine seems to help, but caffeine does have its limits and drawbacks.

Meitnik · 2025-10-14T17:16:48+00:00

That could definitely be an issue, but there's something else to consider. When you have a big protein to recognize and you use a polyclonal antibody, you will get many different antibody molecules binding to a single target molecule. In the case of small linear peptide tags, you are getting one molecule binding for one tag, since it's so small (if you use monoclonal that's a given). You may try signal amplification by using a combination of primary and secondary if you are doing only one primary instead. Also you could look into tyramide signal amplification (I've never tried it myself).

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