| --- Log opened Tue Jul 14 00:00:10 2015 | ||
| fenn | i dont see any fume hood at ccl but there are a couple small rooms if that's relevant and there is access to the ceiling | 00:07 |
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| fenn | looks like they are planning on putting in a fume hood | 00:21 |
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| juul | fenn: you should come by for our tuesday social hangouts. beer and pizza 7 pm | 01:39 |
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| fenn | der.. i'll see if i can make it | 01:51 |
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| kanzure | good | 03:45 |
| kanzure | 23:11 <xentrac> so 1.2MHz/5 gives you a limit of 240 kilobases synthesized per second, at which point you want your conveyor belt to be about 28 million pixels long; at 1200dpi, which is probably optimistic, that's 609 meters, or 6.8 meters per nozzle | 03:56 |
| kanzure | 23:12 <xentrac> (the 28.8 million pixels is 2 minutes) | 03:56 |
| kanzure | hmm apparently people have cnc machined plutonium. i want to do this. | 03:59 |
| kanzure | strange, iran just agreed to be banned from making nuclear explosion simulations | 04:04 |
| kanzure | and also banned from making streak cameras... why would they agree to that. | 04:05 |
| fenn | because nudity is morally reprehensible | 04:21 |
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| fenn | i'm a bit put off by azco's totally lame and incomplete MSDS for phosphoramidites | 04:54 |
| fenn | Potential Health Effects (Acute and Chronic): No data available. -_- | 04:56 |
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| fenn | kanzure i will send that email to counterculture unless you have any additions/subtractions | 05:27 |
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| ebowden | paperbot http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=784816 | 06:09 |
| juri_ | nmz787: is there an open toolchain for programming the ICE40? | 06:11 |
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| CaptHindsight | just out of curiosity why would some want to spend days or weeks to develop new FPGA's, drivers or even an application to replace something that already works really well and costs <$200? | 06:48 |
| CaptHindsight | and only panning on building a few | 06:49 |
| juri_ | someone who has standardized on a different solution? | 06:51 |
| nottimschmidt | < $200 might as well be infinitely expensive for most of the world's population. | 06:52 |
| juri_ | I try to cut costs on every part of the things i build, for the reason nottimschmidt just supplied. | 06:53 |
| juri_ | one of my 3d printers uses a microcontroller salvaged from a battery backup i found on the side of the road. because. | 06:53 |
| nottimschmidt | In the world of collaborative development, fewer barriers to entry means more contributors :D | 06:54 |
| CaptHindsight | nottimschmidt: the <$200 in this example is part of a $10K machine/system | 06:54 |
| narwh4l | It's important to distinguish open design from a hacked design | 06:54 |
| narwh4l | open designs are great for developing countries. Not sure how accessible the microcontroller from a battery backup is to the world | 06:54 |
| CaptHindsight | so if you can't afford the $10K it's not an issue anyway | 06:55 |
| nottimschmidt | CaptHindsight: I don't think this philosophy or way of looking at things should prevent you from doing the work in the way that works best for you. But you should be aware of the motivation of others in reducing cost and complexity everywhere we can. | 06:55 |
| nottimschmidt | Today's $10k machine is tomorrow's $1k machine | 06:55 |
| nottimschmidt | 3D printers are already available in the $300 price range | 06:55 |
| nottimschmidt | That happened one cost reduction at a time :D | 06:55 |
| fenn | it's also easy to say "hey i can buy a printer for $50 so why is this $10k" | 06:56 |
| nottimschmidt | yep | 06:56 |
| juri_ | it's also important to note that today's 3d printer has much better software than yesterday's $1K printer. | 06:58 |
| nottimschmidt | and if that software is well architected, it serves as a library of modular components that makes building the next machine that much easier :D | 07:06 |
| fenn | thanks for your work on transfering things to git btw | 07:07 |
| nottimschmidt | The work I've done with SLS, SLA, and laser cutting was all easier thanks to the 3D printer software available. | 07:07 |
| nottimschmidt | fenn: me or someone else? | 07:13 |
| nottimschmidt | It's been a while since I moved anything to git :D | 07:13 |
| fenn | iirc you moved all the reprap SVN stuff and converted it to .scad files | 07:13 |
| nottimschmidt | Yeah. You have a long memory. :) | 07:14 |
| fenn | it was a big mess because they just threw everything into svn regardless whether it was relevant or not | 07:14 |
| fenn | i think if that hadn't happened nobody would have been able to make improvements to any reprap software | 07:15 |
| nottimschmidt | Thanks for that. Lots of other folks contributed as well. These days I'm thinking a lot about ways to make the firmware and CAD more flexible. Supporting lots of different printable and off-the-shelf tool holders/changers in CAD and the firmware, porting things to javascript, and trying to psych myself into writing a toolpather based on this: http://dspace.mit.edu/bitstream/handle/1721.1/29225/50140264-MIT.pdf?sequence=2 | 07:19 |
| fenn | .title | 07:20 |
| yoleaux | fenn: Sorry, that doesn't appear to be an HTML page. | 07:20 |
| nottimschmidt | 07:20 | |
| nottimschmidt | It's a masters thesis paper that outlines all the algorithms necessary for automatic 5 axis toolpath generation | 07:21 |
| fenn | "automatic 5-axis NC toolpath generation by mahadevan balasubramaniam" | 07:21 |
| nottimschmidt | There's currently no Free software capable of that | 07:21 |
| CaptHindsight | heeks and pycam only do 3-axis | 07:23 |
| CaptHindsight | nottimschmidt: are you going to write a 5-axis CAM app as well? | 07:23 |
| nottimschmidt | CaptHindsight: we'll see. I've been hacking a bit on OpenJSCAD. I like that I can run it on my phone. Most of the world computes with phones. I'd like to teach it to slice and print. | 07:24 |
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| nottimschmidt | I'd like to teach it about multiple tools. | 07:26 |
| nottimschmidt | But I am currently teaching it about CAD libraries. | 07:27 |
| nottimschmidt | So it's easier to make designs from an assembly of pre-built parts, instead of having to build everything from scratch. | 07:27 |
| fenn | is pycam usable in general now? | 07:28 |
| kanzure | fenn: email looks good to me | 07:29 |
| nottimschmidt | We've used it a bit, for the laser cutter. Seems to mostly work, if not fully. | 07:29 |
| nottimschmidt | Several successful cuts. | 07:29 |
| nottimschmidt | This is the tech I'm working toward: http://us.dmgmori.com/products/lasertec/lasertec-additivemanufacturing/lasertec-65-3d#Video | 07:32 |
| nottimschmidt | I can't afford to buy one, but I want one :D | 07:33 |
| fenn | i remember this, very cool | 07:35 |
| fenn | a tig welder could probably do just as well for the additive part | 07:36 |
| kanzure | fenn you might be interested in where nottim isn't working these days | 07:36 |
| fenn | error negative stack size exceeded | 07:36 |
| nottimschmidt | fenn: true. I like the part lifecycle of the powder printing - recycle failed prints in a ball mill, re-print. But a MIG/TIG would work just as well for v1. Good idea. | 07:37 |
| kanzure | http://beacon-center.org/ | 07:37 |
| nottimschmidt | yep, that place. | 07:37 |
| fenn | do you make beacons | 07:38 |
| nottimschmidt | I have been absorbed by the warm and comforting robot bosom of the academic borg. | 07:38 |
| kanzure | evolutionary search algorithm stuff | 07:38 |
| kanzure | nottimschmidt: have i shown you http://verbnurbs.com/ | 07:39 |
| nottimschmidt | Biologists, computer scientists, physicists, ecologists, and so on. Studying computational biology, intelligence, genetic programming, evolutionary algorithms, etc etc etc. | 07:39 |
| nottimschmidt | kanzure: yep! I'm still looking for exactly the right use. | 07:40 |
| fenn | evolutionary algorithms is a big hammer | 07:40 |
| nottimschmidt | yep | 07:40 |
| nottimschmidt | There's a group here doing "On Intelligence" style research as well. Using markov brains, c. elegans models, etc. | 07:42 |
| nottimschmidt | even bigger hammer | 07:42 |
| fenn | i don't know what a markov brain is | 07:42 |
| nottimschmidt | yeah, no one does. | 07:43 |
| kanzure | perfect | 07:43 |
| nottimschmidt | I don't think anything's been published yet | 07:43 |
| fenn | quick, race to the patent office! | 07:43 |
| fenn | "yes hello i'm trying to patent the concept of a markov brain. what is it, you ask? well we don't really know but it sounds cool and we want to prevent anyone else from using it for 20 years" | 07:44 |
| fenn | i think "long short term memory" and "recurrent neural networks" are the new hot topics | 07:45 |
| fenn | basically the same thing | 07:45 |
| nottimschmidt | They're really simple. Imagine an area of memory a few bytes long. Say 8 of the bits in that memory are dedicated to inputs - one bit represents an eye, another bit represents another eye, say one represents a food sensor, etc. And a few more bits represent motor outputs. move forward / back for a left wheel and a right wheel. | 07:45 |
| nottimschmidt | Connect those bits with an evolved network of logic gates. | 07:45 |
| nottimschmidt | Hierarchical Temporal Memories are the shit | 07:46 |
| nottimschmidt | Markov brains can be functionally identical, just another way of expressing the same algorithms. | 07:46 |
| fenn | boolean logic? is there a delay in the transmission from one gate to the next? | 07:46 |
| fenn | in order to make use of rate coding you have to have some kind of accumulator | 07:47 |
| fenn | eh well anyway, i hope it works out | 07:48 |
| nottimschmidt | No delay, but determistic, incremental time. Depends on the implementation, but gates can function as memory, as can bits in the systems memory that are connectable to gates, but don't function as input or output | 07:48 |
| fenn | a long time ago someone did experiments with evolved fpga code, worked beautifully and hardly used any gates at all, but it wouldn't run in simulation or on any other chips | 07:48 |
| nottimschmidt | There are feedback gates in some implementations as well | 07:48 |
| nottimschmidt | Right | 07:48 |
| nottimschmidt | These guys to really interesting things in very few gates as well, but the behavior is deterministic. | 07:49 |
| nottimschmidt | One researcher I know is working on calculating Phi for these things | 07:50 |
| nottimschmidt | https://en.wikipedia.org/wiki/Integrated_information_theory | 07:50 |
| nottimschmidt | Phi's a measure of the amount of information integrated by a system | 07:51 |
| kanzure | .wik | 07:51 |
| yoleaux | Search for an article on Wikipedia | 07:51 |
| kanzure | .wik integrated information theory | 07:51 |
| yoleaux | "Integrated information theory (IIT) is a framework intended to understand and explain the nature of consciousness. It was developed by psychiatrist and neuroscientist Giulio Tononi of the University of Wisconsin–Madison." — https://en.wikipedia.org/wiki/Integrated_information_theory | 07:51 |
| kanzure | bleh | 07:51 |
| kanzure | here's an explanation of consciousness: it doesn't exist. there, problem solved. | 07:51 |
| nottimschmidt | :D | 07:51 |
| nottimschmidt | Prediction is what matters. | 07:51 |
| nottimschmidt | And is how the local physicist defines information | 07:51 |
| kanzure | no really, what predictive benefit does consciousness confer on your models that is impossible without consciousness? | 07:53 |
| nottimschmidt | I'm agreeing with you. :D I think we're memorization and prediction machines, and that's the bulk of what constitutes intelligence. | 07:54 |
| kanzure | i also don't believe in the intelligence word either | 07:55 |
| fenn | they ain't no sich word | 07:56 |
| nottimschmidt | I think that's fair. My understanding is an ecological one, at several different scales. | 07:57 |
| nottimschmidt | Emergent properties of complex interacting systems, all the way down :D | 07:57 |
| nottimschmidt | And all words and names for such things somewhat inadequate and colored extensively by perception | 07:58 |
| fenn | perfect denotation is raw data; all vocabulary carries compression artifacts | 07:59 |
| nottimschmidt | yup | 07:59 |
| kanzure | i'm not complaining about compression artifacts | 07:59 |
| kanzure | i'm complaining about wrong or broken concepts | 07:59 |
| fenn | i don't think consciousness is "wrong" as a concept, it's just "not even wrong" | 08:00 |
| nottimschmidt | kanzure: they are everywhere, and fuck up understanding like a damn fucks up a river. | 08:00 |
| nottimschmidt | brb | 08:01 |
| kanzure | i'm not claiming there are no compression artifacts flying around | 08:01 |
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| kanzure | fenn: email sent? | 08:12 |
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| fenn | sented | 08:18 |
| fenn | oh i should have cc'd you so the replies went to you too | 08:18 |
| fenn | oh well | 08:18 |
| kanzure | well depending on how much you care about that, you can send a follow up where you cc me and say "looping bryan into this" | 08:19 |
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| nmz787_i | juri_: http://hackaday.com/2015/05/29/an-open-source-toolchain-for-ice40-fpgas/ | 09:04 |
| juri_ | nmz787_i: Nice. | 09:05 |
| nmz787_i | CaptHindsight: I'm all for splurging and dropping $$$ when I want to be lazy and not spend a ton of time engineering something... but I also like to have a path forward for thinking of how to decrease costs. I think the others gave lots of good points too. I used to not have lots of $, but still knew that for a lot of things, the parts/materials cost was significantly less than whatever sales pricetag something had slapped on it. $200 doesn't | 09:08 |
| nmz787_i | sound too bad, but I'm pretty sure it could be 1/5th of that or less with some customized boards... or even just picking and choosing from existing boards that are up on ebay, etc... (i.e. ice40 and an easydriver) | 09:08 |
| nmz787_i | CaptHindsight: if for no other reason, I like to have relative comparisons just for judgement and keeping my bearings in a space | 09:09 |
| juri_ | this makes some good sense. it's worth noting the ICE toolchais speaks verilog, and the mesa toolchain is VHDL. | 09:10 |
| nmz787_i | ah, how about the relative size of the fabric in that compared to the mesa? | 09:17 |
| CaptHindsight | juri_: does that even matter? | 09:17 |
| CaptHindsight | seems some people would spend weeks hand packing gates to save $2 since their time must be free | 09:18 |
| juri_ | CaptHindsight: you're right in a fashion. | 09:19 |
| CaptHindsight | if you are going to mass produce it sure | 09:19 |
| juri_ | saving $2 is a big dial if you're expecting people to build hundreds of them. | 09:19 |
| kanzure | heath: you should go hang out with fenn | 09:20 |
| CaptHindsight | 1,000,000 x $2 is a lot of $ | 09:20 |
| kanzure | heath: counterculture labs has 7pm social gathering | 09:20 |
| juri_ | right. | 09:20 |
| nmz787_i | CaptHindsight: those people seem crazy... the largest countries still make $1 a day... so their time is worth more than $2 per weeks | 09:20 |
| CaptHindsight | but cnc glue guns and an inkjet printer are not going to revolutionize their world | 09:22 |
| juri_ | some hackers are very poor, despite being in rich areas. | 09:22 |
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| CaptHindsight | I often see lots of reinventing for the sake of reinventing | 09:25 |
| CaptHindsight | especially with user interfaces | 09:26 |
| CaptHindsight | why do they all seem to get worse? | 09:26 |
| juri_ | we all have radically different UI philosophies. for instance, i prefer writing my code in emacs and building it using Makefiles.. including my 3d models. | 09:34 |
| kanzure | this just sounds like you guys are angry about spending money on stuff | 09:35 |
| kanzure | CaptHindsight: we're investigating counterculture labs as a possible home for the machine | 09:38 |
| CaptHindsight | are they in Oakland or the area? | 09:39 |
| kanzure | oakland | 09:41 |
| eudoxia | anyone here remembers that post in mike darwin's blog where he talked about paying a master glass blower to make a bubble trap | 09:42 |
| eudoxia | i recall it had a tiny insight about the value of apprenticeship | 09:42 |
| fenn | it's all the bay area | 09:42 |
| fenn | oakland is just known as "oakland" because it sucks | 09:42 |
| fenn | and maybe something about a football team... | 09:43 |
| kanzure | "Also, he really cared about the ant hill more than the ants. He would do horrible things to the ants that would encourage them to build a bigger ant hill. (Ants are users, employees were treated very well). As a fictional example, lets say the site was skewing too heavily male by 20%, just add a line in the stored proc that gave a male user a 20% chance of their registration not being written to disk. Voila, site has the proper mix, ... | 09:45 |
| kanzure | ... onto the next problem. He was the most brutally effective person I've ever worked with." | 09:45 |
| kanzure | eudoxia: talked with mike darwin for about 3 hours on the phone the other day. i have prepared for future calls and will record things in more detail. | 09:45 |
| eudoxia | kanzure: cool, what did you talk about | 09:46 |
| kanzure | bottom of http://gnusha.org/logs/2015-07-11.log | 09:47 |
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| kanzure | fenn: ah, heath reports that he will be attending counter culture labs this evening, so perhaps you will run into him if you go | 09:52 |
| fenn | eleitl suggested literally recording the sound of the conversation | 09:52 |
| kanzure | which conversation? | 09:53 |
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| fenn | ugh so much obligation stress.. now i definitely will not be able to get to sleep | 09:53 |
| fenn | i am not going, ok, good. | 09:53 |
| kanzure | obligation stress eliminated by planning to not go? | 09:53 |
| fenn | something like that | 09:53 |
| fenn | i wish it were so simple | 09:54 |
| kanzure | well, you sent the email, if they aren't raging assholes then they should consider the proposal as a group | 09:54 |
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| nmz787_i1 | i just left? | 09:54 |
| fenn | it's basically impossible for me to fall asleep when i know i have to wake up at a certain time | 09:54 |
| nmz787_i1 | oh, no, i am the clone | 09:54 |
| kanzure | fenn: really the root of the problem here is not obligation stress or sleep schedule mismatch but that they are having an in-person meeting (it shouldn't be) | 09:55 |
| fenn | it's not a meeting, it's just beer and pizza | 09:56 |
| kanzure | oh hm | 09:56 |
| kanzure | yeah i guess that's difficult to orchestrate without bodies in the same room | 09:56 |
| fenn | you could use a clever system of catapults | 09:57 |
| kanzure | i think you run out of available pcr barcodes | 09:58 |
| fenn | anyway you should record your phone conversations with mike darwin for posterity | 09:58 |
| kanzure | and if you increase the size of the barcode then the middles of the barcodes start matching the ends of other barcodes because of sequence similarity | 09:58 |
| kanzure | yes, i understand | 09:58 |
| fenn | the length of the oligo is limited anyway | 09:58 |
| kanzure | number of spots is not limited | 09:59 |
| kanzure | xentrac wants a conveyor belt added heh | 09:59 |
| fenn | the barcode is part of the oligo sequence | 09:59 |
| fenn | i dont know what you guys are intending to do with all this dna | 09:59 |
| kanzure | i'm not sure whether to think that single pool gene assembly is going to work | 10:00 |
| kanzure | well, i'd like plasmid and gene assembly | 10:00 |
| kanzure | and genome assembly would be nice | 10:00 |
| juri_ | fenn: replicants. | 10:00 |
| fenn | well it's not really single pool if you're selecting different subsets via pcr and then doing assembly in a separate reaction vessel | 10:00 |
| kanzure | ok, so you pcr-amplify in the same pot, then you extract the ones that you amplified (because most of the extractant is going to be your amplified material)? | 10:01 |
| fenn | ok so how big is a plasmid, 100kB at most right? | 10:01 |
| kanzure | sure 100 kb sounds good to me | 10:01 |
| fenn | well there's orders of magnitude left over if you're doing millions of oligos | 10:01 |
| CaptHindsight | how do you think that they are doing assembly? http://www.adnas.com/products/signaturedna | 10:02 |
| kanzure | this doesn't say anything about gene assembly | 10:03 |
| fenn | "SigNature DNA markers are based on full, double-stranded plant DNA." | 10:03 |
| nmz787_i1 | 100kb is huge for a plasmid | 10:03 |
| heath | fenn: are you planning attending the cc social? | 10:03 |
| nmz787_i1 | more like 40kb max | 10:03 |
| fenn | lol "This botanically engineered solution is shielded by a portfolio of 24 patents, 58 patent applications, and other intellectual property protection." | 10:04 |
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| nmz787_i1 | heh, proxyham http://hackaday.com/2015/07/14/how-to-build-a-proxyham-despite-a-cancelled-defcon-talk/ | 10:04 |
| CaptHindsight | heh yeah | 10:04 |
| fenn | "we applied for so many patents it hurts" | 10:04 |
| kanzure | nmz787_i1: ah didn't know there were 40 kb plasmids. cool. | 10:04 |
| nmz787_i1 | fenn seems to want some proxypizza | 10:04 |
| kanzure | it wasn't a ham | 10:05 |
| kanzure | it was lies | 10:05 |
| kanzure | heath: i have sent you an email that fenn sent earlier to counter culture labs | 10:05 |
| nmz787_i1 | kanzure: hmm, from nets "Yes, the bigger the plasmid the lower the transformation efficiency. However, plasmids up to 200 kb can be transferred by electroporation (hand-on experience). For even bigger plasmids conjugation would be my choice of transfer method." | 10:05 |
| kanzure | cool, glad to hear 200 kb plasmids can get in there | 10:06 |
| nmz787_i1 | kanzure: I don't see that email on the mailing list... was it not sent that route? | 10:06 |
| kanzure | which mailing list? | 10:06 |
| nmz787_i1 | and also "Plasmid stability/ segregation and ease of work. It simply gets more difficult to handle when they are large (> 50 kb)." | 10:06 |
| nmz787_i1 | the counterculture mailing list (google) | 10:06 |
| kanzure | no it was not sent to that email address | 10:06 |
| fenn | i am lame and haven't looked into mailing lists or anything yet | 10:08 |
| fenn | i'd rather talk to a specific person than "hey guyz how bout this thing ... *crickets*" | 10:08 |
| fenn | "All you need to do is cancel the talk and allow tech journos to speculate about National Security Letters and objections to the publication of ProxyHam from the highest echelons of government." | 10:10 |
| fenn | i dont get it anyway... 900 MHz? just use a wifi bridge | 10:10 |
| kanzure | see https://www.reddit.com/r/worldnews/comments/3d7wil/a_project_to_build_a_200_diy_wifi_router_to_help/ct2tvn6 | 10:10 |
| kanzure | i think that making a vitamin a producing probiotic would be a good project to do with this dnajet | 10:13 |
| nmz787_i1 | kanzure might say you could accomplish internet anonymity with a clever catapult setup, throwing zip disks around with encrypted data | 10:13 |
| kanzure | unfortunately that suffers from man in the middle attacks | 10:13 |
| fenn | also disk read head oxidation | 10:14 |
| fenn | as anyone who has tried to read a zip disk knows | 10:14 |
| kanzure | "Sure, I'll plan on being there and speaking with Kathy if I don't see Fenn." | 10:15 |
| fenn | that commenter goes a bit too far calling 900MHz "crap" - it's only 2.6x slower than 2.4GHz and usually you're nowhere near saturating the wifi link | 10:16 |
| fenn | but it does go through trees and fog better | 10:16 |
| kanzure | pick-and-place seems to be the only gene assembly technique available at the moment. you have to limit the number of beads, there's no "assemble as many things as you want simultaneously" technique that anyone has figured out. | 10:24 |
| kanzure | actually, a method that would be nice would be something like: imagine a giant grid where you print all your spots. there are separate groups of spots that will (somehow) be mixed together after initial synthesis. these groups will undergo some chemical reaction for local assembly. then these groups can be mixed together (without pick-and-place), and so on, up to at least 3 or 4 levels. | 10:26 |
| fenn | the trick is to not do it simultaneously | 10:26 |
| kanzure | this could perhaps be achieved by something like, "use a laser to cut some open-top channels between the groups, so that the liquids can mix later, and then you just pcr in the combined droplets" | 10:26 |
| fenn | the pick and place is for error rejection mostly | 10:26 |
| kanzure | it helps for that, but no i think it's so that you can pick which 10 beads you want to run gibson assembly on | 10:27 |
| kanzure | and then you do a 10-bead gibson assembly run in another test tube (or whatever), then you combine the two results and run gibson assembly again | 10:27 |
| fenn | yes the 10 beads that have no errors :P | 10:27 |
| kanzure | quality control is a separate question i think | 10:28 |
| kanzure | the 10 beads would have separate sequences (intentionally) | 10:28 |
| kanzure | with the pcr barcodes | 10:28 |
| kanzure | laser cutting the surface to mix the different groups might work | 10:29 |
| kanzure | plus the same laser could perform laser-assisted-heating pcr | 10:29 |
| kanzure | you could also use stencils to confine different groups, then remove a stencil to allow mixing, and then remove the next stencil to allow the next bracket to fight | 10:30 |
| kanzure | err to allow the next bracket to assemble | 10:30 |
| nmz787_i1 | if you were enriching via the PCR barcodes for a million spots, wouldn't you need a million primers in some storage area | 10:31 |
| nmz787_i1 | ? | 10:31 |
| nmz787_i1 | and I think you could just jet mastermix between the spots and join them together with surface tension | 10:31 |
| kanzure | how big can the spots get before surface tension is not enough | 10:31 |
| kanzure | ah you can add stuff to make surface tension to continue to work i think | 10:32 |
| nmz787_i1 | if you had 4 spots, jet an X of mastermix+enzymes | 10:32 |
| kanzure | hmm | 10:32 |
| kanzure | surfactants | 10:32 |
| kanzure | joining the spots together is an interesting idea | 10:32 |
| kanzure | you would have to lay out your dna bitmap so that you can combine the spots you want in the correct order for assembly, of course | 10:32 |
| nmz787_i1 | well surface tension works for spots millimeters wide (at least thinking of my car window when it rains) so I'm guessing it's only stronger for sub-millimeter | 10:33 |
| nmz787_i1 | for a million spots, how many mers do you need? | 10:33 |
| nmz787_i1 | is that log4(1000,000)? | 10:33 |
| nmz787_i1 | yea | 10:34 |
| nmz787_i1 | so 10-mer | 10:34 |
| nmz787_i1 | 11 gets you 4 million | 10:34 |
| nmz787_i1 | unless the spots are too low conc to amplify later, after ligation and transfer losses.... then I'd say skip the barcodes... rely on hybridization of gibson assembly, etc... then after a few pooled assemblies, amplify with some generic starting barcode and filter by length (by that time there should be an order of magnitude difference between synthesized and amplified) | 10:37 |
| nmz787_i1 | (length) | 10:37 |
| fenn | nmz787_i1: you dont need a million different primers, only however many subsets you have. if it's 10 oligos per assembly then you need 100,000 primers | 10:40 |
| kanzure | moving droplets wouldn't work if you need the wash step to not wash away everything, plus if you're using pores or wells then you need to figure out some other way of mixing between liquid-bridged pores i guess. | 10:42 |
| kanzure | moving/merging | 10:42 |
| nmz787_i1 | fenn: kanzure wants 1.2 millions spots, if they're all unique... won't you want each to have it's own barcode? anyway, if they're destined for assembly, hybridization is kind of the same thing... so do what I said and I think it will be easier | 10:46 |
| fenn | it depends what you're using the barcode for | 10:47 |
| nmz787_i1 | wasn't it supposed to be like a checksum, only good codes hybridize with the primer and amplify? | 10:47 |
| fenn | one system used the barcode to pick out randomly labeled clones of individual oligos. in that case you'd need a million barcodes (or however many oligos you wanted) | 10:47 |
| fenn | another system used a barcode to pick out a subset of oligos for hybridization | 10:48 |
| fenn | er, assembly, not hybridization | 10:48 |
| fenn | it's probably better to use smaller oligos anyway because parallel writes go faster than serial writes | 10:49 |
| fenn | it takes 10 times longer to make an oligo 10 times as long, but less than that to make 10 times as many oligos | 10:50 |
| fenn | like 1.01 times as long maybe | 10:51 |
| nmz787_i1 | maybe you could use 2 barcodes, one at the beginning and one at the end... but then you need to clip them off after amplification and it doesn't ensure there aren't internal deletions | 10:51 |
| nmz787_i1 | which hybridization does help with | 10:51 |
| kanzure | why would you need to clip them off after amplification | 10:51 |
| fenn | to ensure they are random sequences | 10:51 |
| nmz787_i1 | how will your protein function with some repetitive tag every 30 bases or whatever | 10:52 |
| nmz787_i1 | you can't have that | 10:52 |
| kanzure | i think proteins could tolerate that, actually | 10:52 |
| kanzure | especially if it's not an amino acid | 10:52 |
| fenn | er.. every sequence is an amino acid | 10:52 |
| kanzure | hmph | 10:52 |
| nmz787_i1 | i don't know ascii emoticons well enough to describe how I just felt about that comment | 10:52 |
| kanzure | right, right | 10:52 |
| kanzure | i'll go back to writing software | 10:52 |
| kanzure | during pcr and assembly perhaps you don't need wash steps | 10:56 |
| fenn | now that i think about it, it might be difficult to do pcr on a 12"x12" plate | 10:59 |
| fenn | water evaporating and stuff | 10:59 |
| kanzure | neutral atmosphere, humidity could be modified if necessary | 11:00 |
| nmz787_i1 | might need to be a separate machine too, since you don't want water near the synthesis stuff | 11:00 |
| fenn | this is after the plate is removed | 11:00 |
| kanzure | .title http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209801/ | 11:00 |
| yoleaux | Petri dish PCR: laser-heated reactions in nanoliter droplet arrays | 11:01 |
| nmz787_i1 | if you contaminated the inert zone, you'd need to pull vacuum for a while to dry things out | 11:01 |
| fenn | maybe it makes sense to be able to easily break the plate into chips, so you can just put a chip with 10000 oligos on it into a pcr tube instead of messing with a whole plate | 11:02 |
| kanzure | i'm not convinced anyone has a method of assembling a gene from 10k oligos | 11:03 |
| fenn | what was the density again? in oligos per mm^2 | 11:03 |
| kanzure | that was undecided actually. i think at one point we had 100 microns between drops, possibly more. | 11:03 |
| fenn | you're not doing all 10k at a time, only a subset | 11:03 |
| fenn | so 100 oligos per mm^2 | 11:04 |
| kanzure | you could easily end up with 1,000 pcr tubes and now you have a different problem... | 11:05 |
| kanzure | eh i guess 1k pcr tubes isn't the end of the world | 11:05 |
| fenn | the m.laboratorium assembly took roughly that many steps | 11:06 |
| kanzure | yashgaroth says, "Honestly I'd be interested just to leverage it for the isothermal amplification + nicking enzyme project from a while back. Suddenly even a 10mer library doesn't sound too insane if you can get 1000x1000 printing resolution, and the ligase assembly process should be somewhat error-correcting. The longer the fragments, the likelier it is to work." | 11:06 |
| fenn | i didn't understand that at all | 11:08 |
| kanzure | at one point he mentioned in the logs a nicking enzyme method for dna synthesis and gene assembly. | 11:08 |
| CaptHindsight | depending on the surface tension figure ~75um dia spots, and maybe a 25um space so maybe 100um center to center | 11:08 |
| fenn | https://en.wikipedia.org/wiki/Nicking_Enzyme_Amplification_Reaction | 11:09 |
| nmz787_i1 | kanzure: but is yash assuming we're printing the 10mers, or do we still need to store and dispense them? | 11:09 |
| fenn | .c 4^10 | 11:11 |
| yoleaux | 4¹⁰ = 1048576 | 11:11 |
| fenn | why do you need 10-mers for nicking enzyme amplification? | 11:12 |
| nmz787_i1 | that was a different kind of nicking enzyme trick | 11:12 |
| nmz787_i1 | that was from... 2012 I think | 11:12 |
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| * fenn looks at http://gnusha.org/logs/2012-02-05.log http://gnusha.org/logs/2012-02-15.log http://diyhpl.us/~bryan/papers2/DNA/nicking-library-method.jpg | 11:16 | |
| fenn | so this is just to make a crapton of 10mers | 11:18 |
| fenn | like pure white noise | 11:19 |
| fenn | i guess each of these beads would be floating in its own microfluidic water bubble? otherwise what would you do with them | 11:21 |
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| fenn | i hope they're wrong about having to buy 50 liters of acetonitrile at a time | 11:26 |
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| nmz787_i | fenn: the idea was to have a million beads contained individually/separately... then amplify to get a 10-mer, then mix a combo of 10-mers to get your desired sequence | 11:32 |
| nmz787_i | the nicking amplification was so you only have to synthesize the 10-mers once, then you just amplify any time you need one for your desired sequence | 11:33 |
| nmz787_i | and no, they aren't joking about the diluent needing to be purchased in 50L quantities | 11:33 |
| nmz787_i | waste management will be a bitch | 11:33 |
| fenn | i guess i could see the 6-mer thing working if you had one of those programmable electro-microfluidic arrays | 11:35 |
| fenn | contamination would be annoying though | 11:36 |
| kanzure | i'm having trouble keeping track of the performance of known-methods | 11:38 |
| fenn | 1 million individually addressable droplets just seems too hard | 11:38 |
| kanzure | in the microfluidics approach? | 11:38 |
| fenn | in any approach, but especially microfluidics | 11:38 |
| kanzure | or in the micropipetting-at-that-resolution-is-too-weird? | 11:39 |
| kanzure | *is-too-weird sense? | 11:39 |
| fenn | 1 million is a really big number | 11:39 |
| fenn | have you ever seen 1 million of anything? | 11:39 |
| kanzure | does viewing an inkjet printed document count? | 11:39 |
| kanzure | or viewing an lcd screen | 11:40 |
| fenn | i think there are something like 100,000 visible stars in the sky | 11:40 |
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| kanzure | i think another aspect here is that it's all probabilistic anyway | 11:41 |
| kanzure | so.. the only way to get good results is even more quality control :-/ which limits the scale you can do this at anyway. | 11:41 |
| kanzure | unless you have highly parallel quality control | 11:41 |
| fenn | hmm you canhttp://i.imgur.com/cl7Ng.jpg | 11:41 |
| fenn | you can only see 5000 stars in the sky apparently | 11:41 |
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| kanzure | 1k pcr tubes as an output isn't a big deal. i think that would be a good result. | 11:43 |
| kanzure | we can just manually run gibson on martial-arts-bracketed combinations until something interesting pops out at the end | 11:43 |
| fenn | i will smash the plates with my fists until they are really small | 11:44 |
| kanzure | they should be pre-smashed | 11:44 |
| fenn | pyrosequencing can only go up to ~300bp so that should probably be the first assembly size target, which determines everything else | 11:54 |
| fenn | 300bp reads | 11:54 |
| fenn | i dont really have the whole synthesis sequence assembly protocol laid out straight in my head, but i'm pretty sure it involves pyrosequencing at some point | 11:55 |
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| kanzure | this seems like an okay review, http://diyhpl.us/~bryan/papers2/DNA/Large-scale%20de%20novo%20DNA%20synthesis:%20technologies%20and%20applications%20-%20Church%20-%202014.pdf | 11:58 |
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| fenn | even without doing PCR we will want good temperature control to improve "hybridization stringency" (and get the associated error correction) | 12:13 |
| fenn | this implies something like a pcr tube or pcr plate | 12:13 |
| fenn | a laser is not going to cut it | 12:13 |
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| fenn | this is a good paper to read, i recommend anyone participating in this discussion read and try to understand all of it | 12:18 |
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| nmz787_i | fenn: i've got a few million pixels in front of me right now | 12:47 |
| nmz787_i | seeing them all the time | 12:47 |
| nmz787_i | in my pocket | 12:47 |
| nmz787_i | oh, kanzure said it | 12:47 |
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| nmz787_i | http://hackaday.com/2015/07/14/vintage-vinyl-laser-etched-on-a-tortilla/ | 13:17 |
| nmz787_i | kanzure: CaptHindsight here's the old shopping list with some recipe steps at the top https://docs.google.com/spreadsheets/d/1bDsOEVUT5SUXnsP8UowV5iRmUxuk0n_328DqYq_c3J8/edit?usp=sharing | 13:19 |
| kanzure | gevent now has python3 support "officially" (no longer need to use tulipcore) https://github.com/gevent/gevent/issues/38#issuecomment-121001665 | 13:30 |
| kanzure | greenlets and libuv implementation for gevent core loop https://github.com/veegee/guv | 13:31 |
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| kanzure | we should add lots of thermometers everywhere and also some heating elements | 14:13 |
| kanzure | we could do liquid cooling if necessary | 14:14 |
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| kanzure | the los angeles biohackers say that they would be happy to house the machine if necessary, and nmz787 has said yes sorta, so we're good to go i think | 14:14 |
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| archels | I didn't read up fully on what you have been talking about, is there a document/wiki page somewhere? | 14:17 |
| kanzure | gimme an email address | 14:17 |
| juri_ | do we need a wiki? i can fire one up for this. | 14:19 |
| kanzure | -_____- | 14:20 |
| kanzure | juri_: did you know that the wiki has been in the /topic for years now? | 14:20 |
| juri_ | i meant one for this project specifically. ;) | 14:20 |
| kanzure | archels: http://diyhpl.us/~bryan/nucleic/dna-inkjet-qt-07121500.pdf | 14:21 |
| archels | kanzure: who is ONE Labs Inc.? | 14:25 |
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| kanzure | archels: CaptHindsight | 14:27 |
| kanzure | juri_: you probably have not seen the doc either | 14:27 |
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| kanzure | "modular overlap-directed assembly with linkers (MODAL)" http://openwetware.org/images/c/cd/BricksReview.pdf | 14:33 |
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| kanzure | yeast that assemble a collection of oligos into the correct order could be selected | 14:38 |
| kanzure | and then for every large-scale plasmid or genome that you print, you also select a bunch of cells that have mutated enzymatic activity that tends to put the oligos together in the right order, toss the colonies that don't. | 14:38 |
| kanzure | </bruteforce> | 14:39 |
| kanzure | huh the wikipedia article on yeast homologous recombination is.. not good. | 14:44 |
| juri_ | kanzure: nope. ;) | 14:46 |
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| archels | kanzure: sounds cool. What do you want to do with it? | 14:48 |
| kanzure | ligase cycling reaction http://aati-us.com/sites/default/files/Amyris_Inc._Rapid_reliable_DNA_assembly_via_ligase_cycling_reaction_AATI_customer_paper.pdf | 14:48 |
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| kanzure | archels: well i'd like to replace about 30 to 50% of all plant dna on the surface of the planet with synthetic alternatives, and get genome snthesis down to $1/genome or lower | 14:48 |
| kanzure | hm that link claims ligase cycling reaction was able to join 20 dna fragments that were each 1 kb? | 14:49 |
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| fenn | counter culture said yes (see mail) | 15:58 |
| fenn | also note that i am not asleep :\ | 15:59 |
| nmz787_i | how to decide between CCL and L.A.Biohackers? | 16:06 |
| nmz787_i | I think I only know Patrik at CCL... but I know at least Cory and Keoni at L.A.B | 16:07 |
| justanotheruser | Dear Justan Otheruser: I’m writing to acknowledge your message and I will write again when I have more information. Love, NCBI Help Desk | 16:08 |
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| CaptHindsight | hmm first no takers now, two | 16:24 |
| CaptHindsight | we can cut it in half and they can each..... | 16:24 |
| fenn | we can reduce the cost and make ten | 16:26 |
| fenn | a dna printer in every garage! | 16:27 |
| CaptHindsight | recroom | 16:27 |
| CaptHindsight | should we anodize it to look like wood grain? | 16:27 |
| superkuh | https://neurolab.gatech.edu/labs/potter - These guys will sell your email address to spammers. | 16:29 |
| superkuh | That or they have some serious security issues. | 16:29 |
| kanzure | CaptHindsight: i'll try to get some edits sent to you tonight, sorry for the delay, i'm glad we have some potential homes | 16:31 |
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| kanzure | nmz787_i: counter culture labs also has juul (in here) plus ryan bethencourt, patrik dapostraphe, etc. | 16:36 |
| kanzure | ParahSailin: i know you hate all of us for not going with the electrode array, but do you have any tricks up your sleeve for gene assembly? | 16:40 |
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| CaptHindsight | kanzure: print electrode arrays with it | 16:44 |
| kanzure | gene assembly is weird | 16:46 |
| Adlai | you're [a product of] weird | 16:46 |
| kanzure | nottimschmidt: are you familiar with yeast homologous recombination? when attempting to assemble genomes from oligos, we should select yeast that put the oligos in the correct order better. but this unfortunately requires dna sequencing and yeast selection.. | 16:47 |
| kanzure | (dna sequencing is to measure the quality of the yeast's work) | 16:47 |
| kanzure | .title http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107329 | 16:56 |
| yoleaux | PLOS ONE: A Rapid and Simple Method for DNA Engineering Using Cycled Ligation Assembly | 16:56 |
| nmz787_i | kanzure: a main idea I had was to use expression as a checksum... always make GFP, and discard any transformants that don't glow | 16:58 |
| nmz787_i | or rather, only pick the glowing ones | 16:58 |
| kanzure | "Gibson Assemblies were designed for megabase-sized DNA sequences with hundreds of base pairs of homology, but have been adapted to the kilobase and smaller scale of common laboratory applications such as protein engineering [9]. Gibson Assembly has limitations too, such as increasing inefficiency when the number of inserts increases, inability to assembly small (<100 bp) sequences, and complex and error-prone addition of homologous ... | 16:59 |
| kanzure | ... sequences that are needed to direct the orientation and order of the assembled product." | 16:59 |
| kanzure | nmz787_i: yep, agreed. | 16:59 |
| nmz787_i | bbl | 16:59 |
| nmz787_i | /me gone FIBin | 16:59 |
| kanzure | "Thermostable polymerases were the key to PCR, and similarly thermostable ligases [10] have enabled applications based on a cycled-ligation reaction (CLR) [11]. Cycled ligations have been used for a variety of purposes [12]–[15], such as synthesizing genes using synthetic oligonucleotides [16], to directing blunt-end ligation reactions using linker oligonucleotides [17]. Notably, the high specificity of CLRs enables the detection of ... | 16:59 |
| kanzure | ... SNPs using a Ligase Chain Reaction (LCR) [18]. Here, we present a DNA fragment assembly method based on a LCR. We use short 40 bp Scaffold Oligonucleotide Connectors (SOCs) that enable directed, scarless, in vitro assembly of multiple DNA fragments into a transformable plasmid in a single reaction. SOCs can be re-used in alternative assembly designs. We apply these cycled ligation assemblies to construct DNA products containing many, ... | 17:00 |
| kanzure | ... variably sized, inserts. These cycled ligation assemblies are efficient and easy to design and run." | 17:00 |
| kanzure | i wonder if you have to increase the amount of time you wait for ligase to work as you increase the number of cycles, because otherwise ligase is less likely to bump into the correct location for the larger fragments | 17:01 |
| kanzure | "The molar concentration ratio of SOCs to insert strands proved critical. Too few SOCs in the reaction made early assembly events unlikely, while too many SOCs interfered with assembly in later cycles. With a 5:1 molar ratio, optimal SOCs are needed only to initiate the assembly reaction. Once opposite strands are ligated, they serve as templates for assembling additional complementary strands. In early cycles SOCs were required to ... | 17:03 |
| kanzure | ... initiate the assembly of the complementary strands (Figure 1A–D). In later cycles (Figure 1E), if SOCs are bound to already-ligated strands they compete with productive assembly of complementary strands. The 5:1 ratio (Figure 1F, lanes 4) is the optimal balance between these conflicting requirements." | 17:03 |
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| kanzure | "Easily designed Scaffold Oligonucleotide Connectors (containing the last 20 bp of the first sequence and the first 20 bp of the next) guide and direct the assembly, but are not incorporated into the final product. The same amplified starting fragment can be reused from one DNA assembly plan to the next, with only the SOCs changing to guide alternative constructions. A given sequence (such as for GFP) need only be amplified once to be ... | 17:06 |
| kanzure | ... assembled into different locations in any number of constructs. This is in stark contrast to comparable assembly strategies, where each insert must encode its own assembly instructions through attached restriction sites or homologous sequences, hampering the reusability of a sequence from one assembly reaction to the next." | 17:06 |
| kanzure | "The specificity of CLAs even allows multiple distinct assembly reactions to take place simultaneously in the same tube. Inclusion of a vector backbone in a CLA enables the direct transformation of assembled circular constructs into competent cells immediately following assembly. However, CLAs are not ideal in all situations. Due to cycles of denaturation and annealing, highly homologous insert sequences can interfere with each other’s ... | 17:07 |
| kanzure | ... assembly, reducing efficiency. Gibson assemblies share this limitation." | 17:07 |
| kanzure | ok, well it sounds like this method would benefit from inserting some "junk dna" once in a while to attempt to prevent highly homologous sequences | 17:08 |
| kanzure | or maybe using unnatural nucleotides could help here. | 17:08 |
| kanzure | "The efficiency of CLAs decreases as the size of the assembled DNA fragments grows above 5–6 kb. This effect is likely due to the probabilistic nature of CLAs, where productive assembly is due to four-part interactions between two single stranded DNA fragments, a SOC, and Taq ligase. As the length of DNA fragments increases, these rare events are crowded out by the higher probability of off-target binding between various combinations ... | 17:09 |
| kanzure | ... of SOCs and DNA fragments." | 17:09 |
| kanzure | well, increase the time then.... duh? | 17:09 |
| fenn | what does a "cycle" consist of? | 17:09 |
| kanzure | "At the scale of the most common DNA assembly goals, using fragments from <500 bp to 5 kb, cycled-ligation assemblies are efficient and powerful, able to assemble many inserts in a single reaction." | 17:09 |
| kanzure | figure 1a | 17:10 |
| kanzure | figure 1a shows a cycle | 17:10 |
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| fenn | so its just raising the temperature to 95C, then letting it cool to 60C to be ligated | 17:13 |
| kanzure | also in addition to increasing the time or duration during the later cycles, you could also decrease the volume of the liquid by various forms of trickery, which should increase the probability of the correct events happening | 17:14 |
| fenn | i guess i figured this was how it worked and why would anyone do it differently | 17:16 |
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| kanzure | i think most people synthesize some stuff at the start and end of each strand, and then they hope polymerase will match them up | 17:17 |
| fenn | there was something about "chewing back" to expose matching ends | 17:17 |
| fenn | but that wouldn't leave a scar | 17:17 |
| kanzure | the downside of cycled ligation assembly is that all of the oligos in all the spots need to be converted to dsDNA first, and then also there needs to be some printed extra oligos to serve as the connectors | 17:18 |
| fenn | yeah you don't end up saving any base pairs because you have to print the connectors too (if saving on base pairs were the point, which it probably isn't) | 17:18 |
| fenn | i don't think it needs to be converted to dsDNA first | 17:19 |
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| fenn | maybe that's the point of this method; it doesn't need to be dsDNA | 17:21 |
| kanzure | "A thermostable ligase (green) specific for nicks in dsDNA seamlessly ligates the two bottom strands. (E) Subsequent cycles of denaturation and annealing/ligation allow the thermostable ligase to ligate the top strands, using the ligated bottom strands as a template. Further cycles of ligation exponentially increase the amount of correct product as the products of early rounds served as an expanding pool of template for later cycles." | 17:22 |
| fenn | the connector makes it dsDNA for like 40bp | 17:22 |
| fenn | anyway you want to make dsDNA from your oligos so you can reject mismatches due to errors | 17:23 |
| fenn | trying to cheap out and overlap only the minimum lets errors sneak through in the parts with no overlaps | 17:24 |
| kanzure | right, right, bolting on some quality control to the original synthesis would be nice as well | 17:27 |
| fenn | that's what i was talking about | 17:27 |
| kanzure | maybe some fluorophores can be purchased attached to the phosphoramidites or something | 17:28 |
| kanzure | cory just suggested the same "place a drop of water/solvent near the spots that you want to combine, then grab via pipette" idea a few moments ago. | 17:34 |
| kanzure | he also suggests using a photocleavable linker and using a laser or dmd to select which spots of interest you want to pick up | 17:34 |
| fenn | you'd want orange-colored plexiglass in the enclosure then | 17:35 |
| kanzure | so we still don't have imaginary number wavelength lasers? | 17:37 |
| kanzure | where's our physicist | 17:37 |
| fenn | we do, but the light they emit travels a right angles to reality | 17:37 |
| fenn | the "place a drop of water" is equivalent to "wall-less wells" based on surface patterning/coating of the glass slide with rain-x | 17:38 |
| kanzure | right | 17:39 |
| fenn | this is good because it lets you keep droplets separate but you don't have walls in the way of the print head | 17:40 |
| fenn | however it doesn't let you do PCR right on the plate, is this a problem? | 17:40 |
| fenn | stupid water | 17:41 |
| kanzure | which one doesn't let you do pcr? | 17:41 |
| fenn | s/PCR/stringent hybridization/ | 17:41 |
| fenn | having no walls | 17:41 |
| fenn | the water would just evaporate from the droplet at 95C | 17:42 |
| kanzure | ah right this is why the other stuff was in an oil bath | 17:42 |
| kanzure | hmph | 17:42 |
| kanzure | well look- at some point, the output matter does have to get off of the surface and into at least 1 tube | 17:42 |
| fenn | harrumph | 17:43 |
| kanzure | was the goal to avoid pipetting tiny drops and tiny pores? | 17:44 |
| fenn | you want to do stringent hybridization as early as possible | 17:44 |
| fenn | the goal was to reduce the amount of pipetting | 17:45 |
| kanzure | for some reason i keep forgetting that gel extraction is a thing that is supposed to work | 17:45 |
| fenn | it's a pain though | 17:45 |
| fenn | why the hell do people use gels | 17:45 |
| fenn | there should be a commonly available inexpensive DNA length spectrometer | 17:46 |
| fenn | just a chromatography column based on gel electrophoresis | 17:46 |
| fenn | then at the end you have a UV LED and photodiode to detect the dna coming out | 17:47 |
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| fenn | Viper168: hey are you the laser guy | 17:47 |
| CaptHindsight | a laser to photocleave? | 17:49 |
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| CaptHindsight | what spot size and what wavelength does it require? | 17:50 |
| kanzure | i don't understand "you don't have walls in the way of the print head" | 17:50 |
| kanzure | CaptHindsight: still deciding if it's necessary | 17:50 |
| fenn | the drops exit the print head at some angle close to 90 degrees but not exactly 90 degrees... the farther away from the surface you are, the more position error you get in where the drops land | 17:52 |
| fenn | so the head should be as close as possible to the surface for best accuracy | 17:53 |
| CaptHindsight | it'll be around 1mm | 17:53 |
| CaptHindsight | the drops also need to form in flight | 17:54 |
| CaptHindsight | it also makes a difference if the head is stationary or moving | 17:54 |
| kanzure | they wont be drops unless it's liquid- the default azco phosphoramidites are solid | 17:55 |
| fenn | its a brown bottle with 1 gram of solid in it? | 17:55 |
| kanzure | hrmmm | 17:55 |
| CaptHindsight | what vehicle is used in the liquid versions? | 17:56 |
| fenn | it might make sense if the goal is to prevent water contamination | 17:56 |
| CaptHindsight | solid block, powder, goo? | 17:56 |
| fenn | vehicle? | 17:57 |
| fenn | i assumed the solvent was acetonitrile | 17:57 |
| CaptHindsight | solvent/vehicle, liquid carrier | 17:57 |
| fenn | thinking about running this thing... if it just dumps a stream of acetonitrile on the plate with each pass, that's going to create a lot of waste solvent | 18:01 |
| fenn | it seems like a fog gun or high pressure sprayer would use much less solvent | 18:01 |
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| fenn | but it's a sealed box so you have to draw "air" from inside the box | 18:02 |
| CaptHindsight | 3-methoxypropionitrile | 18:05 |
| CaptHindsight | 2-methyl glutaronitrile | 18:05 |
| fenn | why is it different | 18:05 |
| fenn | acetonitrile has relatively low toxicity compared to i.e. propionitrile | 18:06 |
| CaptHindsight | have to see how much solvent wash is really needed | 18:07 |
| fenn | how did posam blow-dry the slides even though it was a sealed container? | 18:08 |
| CaptHindsight | the drying nozzles are next to the printhead | 18:09 |
| fenn | but what kept the enclosure from turning into a balloon | 18:10 |
| CaptHindsight | and they also scrub the gas inside for moisture | 18:10 |
| CaptHindsight | "The reagents are currently removed from the slides by an inert gas stream. Increasing the size of the stream or replacing it with a differentmechanisism should be investigated" | 18:12 |
| CaptHindsight | fenn: positive pressure inside the enclosure with a check valve to outside atmosphere | 18:16 |
| CaptHindsight | the POSaM manuals are not the best write up, but certainly not the worst | 18:18 |
| CaptHindsight | wish they would have included more pics or had a demo video of operation | 18:19 |
| CaptHindsight | I could have skipped trying to read their minds in certain sections | 18:19 |
| kanzure | fenn: juul: heath says he is at CCL staring awkwardly at three other people that don't seem to be fenn or juul | 18:19 |
| kanzure | CaptHindsight: posam was built before the invention of youtube. ouch. | 18:19 |
| CaptHindsight | and thankfully twitter | 18:20 |
| fenn | lol ok i guess i will head over to ccl then | 18:20 |
| fenn | eta 35 minutes | 18:20 |
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| kanzure | fenn: let me check with him first | 18:21 |
| CaptHindsight | hasta | 18:21 |
| CaptHindsight | going back to reading People | 18:21 |
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| kanzure | is that how you use your down time? :-) | 18:21 |
| CaptHindsight | down time for me is usually passing out | 18:22 |
| kanzure | fenn: whoops i don't know a current phone number for him. i sent him an email (and cc'd you) instead. | 18:23 |
| fenn | old? 256 274 4225 new? 347 430 7406 | 18:27 |
| kanzure | old: 256 274 4225 and 256 740 2037 | 18:30 |
| kanzure | no answer on 347. whatever. | 18:34 |
| ParahSailin | dunno | 18:54 |
| kanzure | ParahSailin: opinion on the ligase protocol in http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107329 ? | 18:58 |
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| ParahSailin | ligase doesnt usually misbehave | 19:10 |
| kanzure | .wik dna ligase | 19:15 |
| yoleaux | "In molecular biology, DNA ligase is a specific type of enzyme, a ligase, (EC 6.5.1.1) that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond." — https://en.wikipedia.org/wiki/Dna_Ligase | 19:15 |
| ParahSailin | at least you're trying something | 19:17 |
| kanzure | hm? | 19:20 |
| kanzure | .wik ligation (molecular biology) | 19:21 |
| yoleaux | "In molecular biology, ligation is the joining of two nucleic acid fragments through the action of an enzyme. It is an essential laboratory procedure in the molecular cloning of DNA whereby DNA fragments are joined together to create recombinant DNA molecules, such as when a foreign DNA fragment is inserted into a plasmid." — https://en.wikipedia.org/wiki/Ligation_(molecular_biology) | 19:21 |
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| fenn | no sign of heath here | 20:20 |
| kanzure | oh i thought i had stopped you in time | 20:40 |
| kanzure | "students who try a homology-based gene assembly method have a high probability of success on the first try" http://www.biomedcentral.com/content/pdf/s13036-015-0006-z.pdf | 21:00 |
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| nmz787_i | fenn: they likely invented chemical synthesis before finding/mutating a thermostable ligase | 22:49 |
| nmz787_i | so the FIB wizard said if I make some trenches in bulk silicon that backed up next to each other, then turn the wafer sideways to pierce through the wall separating the two, that he could probably make a 60nm hole. Piercing through gold leaf or some other metal leaf would be easier and he could get a lot smaller (because it's thinner, so less redeposition) | 22:51 |
| nmz787_i | but that the leaf would disintegrate with surface tension most likely | 22:51 |
| nmz787_i | another idea I had was making he same trenches, but then connecting the two with a nano trench... such that it acted the same as a pore/channel with a cover-slip on top | 22:52 |
| nmz787_i | but the wizard said that he thought getting the cover slip on top would be troublesome | 22:53 |
| nmz787_i | kanzure: that ligase technique is essentially what I was thinking, except I thought to make the SOCs the same length as the other fragments, since the starting oligos wouldn't be dsDNA anyway. | 22:56 |
| nmz787_i | well, I guess I also thought that using the adapters up would be a good thing as far as keeping the reaction zone tidy | 22:57 |
| nmz787_i | fenn: they used different solvent than acetonitrile because they said it made efficiency increases | 22:57 |
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