This kept me awake. I must know.

Is it possible to create an AI from clockwork?

I'd imagine collisions would be a fairly normal thing without some centralized body approving paths of orbit.

Previous thread here

I have an update! It turns out it wasn't false positives, contaminated glassware, volumes, antibiotic levels, or even the wrong plasmid. Turns out the strain just wouldn't carry the plasmid consistently for some reason. Switching to another strain helped. A handy little imgur album showing some pictures of the problems and solution can be found here

I am trying to grow HMC-1 cells, and have been passaging them into new flasks after spinning down the culture. But how do i avoid the cell debris? I would also like to know how to check the viability of the cells.

It seems to me that collecting and storing everyone's phone calls and emails would be extremely hard and that including video would be even harder due to the difficulty of putting all of that data on physical media and then backing it up. Is it easier than it seems?

I am in the middle of what has been a long an arduous fight with cloning. I got a bunch of cDNAs from the Human Orfeome library, all of which are in a vector designed for gateway cloning. My lab doesn't do gateway, so I made primers and PCR'd out the products.

The PCR primers look generally like this, and are going into a vector with an N terminal GFP.

fwd: NNN - EcoRI site - ATG - GGG TCC GGA (a GSG linker) - NNNNN (complementary site).

(The Kozak is at the start of the EGFP, so I did not include one here).

Rev: NNN - BamHI site - NNNNNN... (rev complement)

The complementary sites are 18 bp total for the primers. I have checked and they do not seem to include any restriction sites I am using. They also would be in frame as the GSG linker is in sets of 3.

I get PCR product at the correct size, I PCR clean up using Qiagen buffers and 3rd party columns (I have used uPrep and Zyppy). I then digest using fermentas fast digest enzymes for 45 minutes at 37 degrees C, then run them on a gel, cut and gel purify. I try and keep the exposure to UV as quick as possible.

I also cut the backbone with fast digest enzymes for 45 minutes and purify in the same manner. I usually do a 40ul digest with 5 ul of plasmid or less.

I ligate either 1 hour on the bench with T4 ligase or 18 degrees C overnight. We use individual alliquots of ligase buffer and the ligase is fairly new.

I transform using heat shock in DH5alpha and plate, depending on the backbone concentration and how long I digested I could either get just a handful of colonies or many. I stopped doing control plates when I started doing colony PCR, but I did find that I would often get less colonies on the plate that had insert vs the one that didn't... I assumed singly cut insert was competing for the cut backbone in that case.

So yeah, I re-streak out colonies on a new amp plate and then I do colony PCR on them with a FWD primer from my vector and a reverse primer from my insert... sometimes I get hits, and then I test digest.

Long story short, I never get the right product. It's driving me crazy. I don't get vector back, but I get weird ass bands.

I know my restriction enzymes are working because I have done other digests to check. I know my backbone is right because I've done test digestions on it. I know my PCR products are at the right size.

What cloning gremlin have I been infected with? :<

Here is an example of today's thrilling result

The gel with the numbered lanes 1-20 are colonies from a transformation, which were then picked and digested as in the gel with only 4 lanes + marker. It should have run as one vector backbone band and one insert band at around 500.

TL;DR cloning isn't working, no idea why, I'm not just getting re-ligated backbone, I'm getting completely weird products.

Yesterday, SpaceX made history by successfully launching their previously used Falcon 9 first stage. This accomplishment most certainly means a great deal for the future of space travel, as it allows the cost of future launches to go down drastically by reducing or eliminating production time and materials.

Feel free to discuss the launch here, or feel free to link other relevant discussions from around the web.

I am taking a microscopy class at my university and have access to a Nikon Eclipse TE2000-S. What are cool things I can do? Any self diagnosis I can do on myself? Not as interested in "cool pictures" as I am in things that I could actually analyze data from but I'm open to all ideas.

I also have 24 access to an Instron E1000 materials testing machine, probably less useful for stuff I'd be interested in but I figured I'd put that out there in case it sparks any ideas in people.

The point of me having access to these machines is so I can learn how to use them in every way I can, so I am very much allowed to do anything as long as it isn't illegal or harmful to the university. In fact: I think me doing things independently would be encouraged.

EDIT: I'm now starting a project tracking endosome speed in LLCPK cells, for anybody who wanted to know the thrilling conclusion of all of this.

As the title states, I have bacterial transformations growing in plates, but when I inoculate liquid cultures from the plates, I get no growth. Restreaks from the plates also grow on additional plates. The cultures, inoculated at ~6pm, are still clear at 10am the following morning with a few sticky white clumps (dead, lysed bacteria, from my understanding). It's worth while to mention that the plasmid is AmpR marked, and we use Carb plates and liquid media--in part this important, since it's my understanding that AmpR is secreted, so the Carb/Amp content of a liquid culture is essentially all degraded following a period of time. As a result, liquid culture should be more permissive than plates. The plates I used for plating are a few weeks old (stored at 4º in foil), but a negative control (transformed with water) showed no growth on the plates, so I'm hesitant to think these are false positives resulting from degraded Carb. I've tried using Carb at 100ug/mL (the concentration on the plates) and 50ug/mL with the same effects (no growth, some dead clumps). I'm trying growing some overnights tonight in LB without Carb to see if I get growth at all--I made a mock overnight of just LB to guard against false positive growth misleading me. The bacteria used for inoculation were only on the plates a few days, so I don't expect that they're dead... but I'm at a bit of a loss. Thoughts? Ideas?

TL;DR: newly plated AmpR bacteria on non-degraded Carb plates show no growth in inoculated Carb overnights. Halp?

I have a number of FCS file that I need to analyze and the software that generates them is on one (very heavily used) computer. Anyone have any suggestions for free flow cytometry analysis software? I am currently using flowCore in R but wouldn't mind something more GUI based

Like the post says. I'll be operating an AFM and haven't had experience before. I've read up on the basics of how it works and I've been shown how to operate the machine, but I was just wondering if there was anything else out there that I'm missing. Sorry if this isn't the correct subreddit; I just found out about /r/askscitech.

Hi SciTekkit, MSc student here... I've got some RNA work coming up, probably in few months, and am not too keen for the protocol my lab uses (suffice to say that chloroform and hot phenol feature heavily). So does anyone know of another protocol? Not a kit-set, less because I'm idealogically opposed to them, and more that pragmatically my supervisor might go for a new method, but only if it's cheap and mildly dangerous :) Danke

What problems do we have now that computers will solve in the coming future?

We’ve had projects like Seti@Home & Mapping the human genome, Im curious what do you guys think we will see in the coming future for giant problems that we will solve with our massive amount of computational power we are amassing as a species? Youtube gets over 500 hours of video content created every minute. This massive amount of audio and video data, combined with giant increases in GPU rendering capabilities have made things like DeepFakes possible.

What problems do we have now that computers will solve in the coming future? Big number problems like the 3 Body Problem, or N+1 come to mind. I’ve heard quantum computing will make finding large prime numbers a snap. Threating Internet cryptography due to piracy.

Which, speaking of piracy, torrenting or illegal file sharing has made it not only possible, but completely free to acquire every song ever recorded. That is an insane thought that people 30-40 years ago never had access to. A free library of every movie ever made. Think how that contrasts to even the idea of a video store.

So how is having access to all these thinking machines going to change how humans function in a society?

Will we map every genome? Create an AI that not only creates video games on the fly but also is always better than us at playing it? What sort of crazy things are in the pipe?

Seeing all these new and innovative technologies just makes me wander, will we ever one day run out of new ideas or new ways to innovate previous technologies to be any more efficient?

I realize the inherent problems in this but I do feel like it should be feasible. It would be nice to be able to go outside anywhere and get a full glimpse of the real night sky. I'm sure there could be some kind of goggle that has a simulated reality of what the sky SHOULD look like by estimating your location through gps...but is there anyway to just filter the light out?

I've used Weeder Web 2.0, and it was OK, but the site seems to be down. I'd love one that shows any possible new sites, as well as established sites, like E boxes or Forkhead binding sites. Are there any somewhat easy to use sites? Thanks!

In the superconductor Wipeout video that's going around, they show a researcher using a handheld device that appears to store liquid nitrogen in a small cylinder. It looks exactly like the sort of thing you'd use with <28g CO2 or N2O cylinders, and shows up around 00:25 clearly captioned as "liquid nitrogen".

The thing is I've never seen LN2 packaged like this, and I don't think it's possible at room temperature without ridiculously exotic (and possibly purely theoretical) materials.

I assume they're just using a CO2 canister as a prop for the video, but I'm wondering if any real cryonauts can comment.

Edit: it does appear that the whole video was in fact a hoax/viral ad! Pretty good CG tho; I didn't notice anything weird about the cars/track/smoke trails till I watched the 720p version.

I was wondering if anybody here could give some advice about a going into career as a research lab tech. After graduating from college I worked in a lab for a few years, which I loved, then went into an MD/PhD program, which I absolutely hate. I'm thinking hard about dropping out and going back into a position as a tech/lab manager. The appeal of a (mostly) 9-5 job without the stress of call/grant writing where I get paid to do what I love sounds extremely appealing to me. However, the obvious consequences of never being a PI/physician and the enormous difference in pay are making me hesitant. Also, I only have a bachelors degree, which might limit my options in career advancement. Right now I'm in no debt and I'm not under any financial obligation to finish the program I started. Do any career techs want to chime in on this and their experiences? How stable has your career been? Do you ever find yourself tired of watching students come and go as you stay in the same place? Does the work stay interesting and engaging year upon year?

Hi all,

Just started a new research job and am relying a lot on metal sputtering processes. I've sputtered metal coatings in university, but now I have to design my own procedures and am a little lost. Being a native English speaker working in non-English speaking country does not help the current situation very much--to say the least.

Having studied electrical engineering in university, I didn't get much of a chance to dive deep into the theoretical or practical backgrounds related to sputtering. I've read a few papers about how Argon pressure, substrate temperature, and deposition power can affect the crystal structure of sputtered metals and have tried out a few different conditions. However, I'm left with horribly oxidized coatings (I have no clue as to how) and/or large grains (up to 1 um when target deposition was only 700 nm) with voids between them creating a very uneven surface.

Do any of you have experience sputtering metals? Could you kindly point me in the direction of some good textbooks or background information I could read up on to help me fine tune my processes more quickly? One of the biggest problems is that a lot of people use our sputtering equipment and I'm lucky to sputter a new batch of trials once a month. Plus getting a good idea of what metal systems work well together or don't work well together along with general theory would go a long way. And rules of thumb are always welcome! Things like "don't sputter Ag on a Si substrate without an adhesion layer even if you think it won't work and especially if your friend tells you it will."

Thanks!

I'm using both the Millipore EZ-ChIP kit (CAT# 17-371) and have tried making my own reagents, and I can't even get the positive control, RNA polymerase II to work in either ES, EB-differentiated, or MEF cells.

The sonication is working well and the bands are showing up between 200-1000 BP, which I've read is perfect for PCR analysis of ChIP. We hope eventually to do ChIP-seq, which requires smaller fragments (~250), but it seems like this is just a matter of increasing sonication parameters.

I've measured the DNA concentration at several points during the protocol, and the DNA essentially disappears between the pull down and elution stages (since the DNA is bound to agarose beads, I can't measure the concentration reliably in between these stages). I don't know if the antibodies aren't working, the DNA is simply getting degraded, or the elution from the beads isn't working.

EDIT: If anyone finds this, it looks like it was the primer that was 3kb down from the promoter region where RNAP2 binds. Noob mistake. DNA concentration does drop dramatically after the pulldown, but that is to be expected since relative to total DNA, immunoprecipitated DNA will be low in concentration.

We've been having problems with our columns and my advisor has asked me to look at our sigma and tau values, but I have no idea what information to gleam from them.