| --- Log opened Sat Jul 18 00:00:14 2015 | ||
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| JayDugger | Good morning, everyone. | 00:44 |
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| JayDugger | Anyone remember a paper describing the effects of tDCS on the likelihood of lucid dreaming? | 01:53 |
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| JayDugger | Don't all answer at once. | 06:11 |
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| kanzure | JayDugger: all i gots is http://diyhpl.us/~bryan/papers2/neuro/tdcs/ | 06:53 |
| QuadIngi | kanzure, hey I might do some research/personal testing on tdcs, I'll make sure to share it when I'm done | 06:54 |
| kanzure | i didn't know that there was video of the fox domestication work https://www.youtube.com/watch?v=0jFGNQScRNY | 06:55 |
| QuadIngi | Hey, what's the purpose of the oxygen equipment you were talking about setting up a while back (six months?) kanzure | 06:57 |
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| kanzure | oxygen equipment. hmm. | 06:58 |
| FourFire | something about gluing something in a vacuum | 07:02 |
| JayDugger | Thank you both. I haven't yet searched the archive, and it doesn't look likely to happen today. | 07:06 |
| CaptHindsight | 1) the first base gets a silane bond to the substrate. This bond is cleaved after synthesis with a free 3'-hydroxyl | 07:09 |
| kanzure | here is a checklist for chemistry projects http://safety.uchicago.edu/files/Lab%20Design%20Guide%20Checklist.pdf | 07:10 |
| CaptHindsight | The starting material is the solid support derivatized with the nucleoside which will become the 3'-hydroxyl end of the oligonucleotide. | 07:11 |
| CaptHindsight | the nucleoside is bound to the solid support through a linker attached at the 3'-hydroxyl. The 5'-hydroxyl is blocked with a dimethoxytrityl (DMT) group | 07:11 |
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| CaptHindsight | 2) SYNTHESIS The first step of the synthesis cycle is treatment of the derivatized solid support with acid to remove the DMT group | 07:12 |
| CaptHindsight | This frees the 5'-hydroxyl for the coupling reaction. | 07:12 |
| CaptHindsight | An | 07:13 |
| CaptHindsight | activated intermediate is created by simultaneously adding the phosphoramidite nucleoside monomer | 07:13 |
| CaptHindsight | and tetrazole, a weak acid, to the reaction column. The tetrazole protonates the nitrogen of the | 07:13 |
| CaptHindsight | phosphoramidite, making it susceptible to nucleophilic attack. This intermediate is so reactive that addition is complete within 30 seconds. | 07:13 |
| CaptHindsight | the phosphoramidite is blocked at the 5'-OH with the dimethoxytrityl group. | 07:13 |
| CaptHindsight | The next step, capping, terminates any chains which did not undergo addition. Since the unreacted chains have a free 5'-OH, they can be terminated or capped by acetylation. | 07:14 |
| CaptHindsight | Capping is done with acetic anhydride and 1-methylimidazole.5 Since the chains which reacted with the phosphoramidite in the previous step are still blocked with | 07:14 |
| CaptHindsight | the dimethoxytrityl group, they are not affected by this step. Although capping is not required for | 07:14 |
| CaptHindsight | DNA synthesis, it is highly recommended because it minimizes the length of the impurities and thus facilitates product identification and purification | 07:14 |
| CaptHindsight | The internucleotide linkage is then converted from the phosphite to the more stable phosphotriester. | 07:15 |
| CaptHindsight | Iodine is used as the oxidizing agent and water as the oxygen donor. This reaction is complete in less than 30 seconds | 07:15 |
| CaptHindsight | After oxidation, the dimethoxytrityl group is removed with a protic acid, either trichloroacetic or dichloroacetic acid. The cycle is repeated until chain elongation is complete. | 07:15 |
| kanzure | and yet no washing steps were mentioned | 07:16 |
| kanzure | nobody finds this strange? | 07:16 |
| CaptHindsight | The oligonucleotide is cleaved from the support by a one-hour treatment with concentrated ammonium hydroxide. | 07:16 |
| CaptHindsight | it's mentioned later | 07:19 |
| ebowden | What're you reading from? | 07:19 |
| CaptHindsight | I really want to send the writers to an editing class | 07:19 |
| kanzure | ebowden: http://diyhpl.us/wiki/dna/synthesis/notes/ | 07:19 |
| ebowden | Thanks. | 07:20 |
| CaptHindsight | Immediately before detritylation, the CPG support is washed with acetonitrile to eliminate traces of the preceding reagent. | 07:20 |
| CaptHindsight | Any residual TCA must be removed by an acetonitrile wash | 07:20 |
| CaptHindsight | to prevent detritylation of the incoming phosphoramidite. If the phosphoramidite monomer becomes | 07:20 |
| CaptHindsight | detritylated, an unwanted dimer can form in solution and then couple to the support-bound | 07:20 |
| CaptHindsight | nucleotide chain. Continued chain propagation would result in some sequences being longer than the product, making purification difficult. | 07:20 |
| CaptHindsight | Following both acetonitrile washes, the remaining solvent is forced out of the column by an argon | 07:21 |
| CaptHindsight | reverse flush - argon passes through the column from top to bottom and pushes the liquid to waste. | 07:21 |
| CaptHindsight | so they wrote the steps in order more than once going over the different details in different paragraphs vs just going through the steps one by one and including the details as they go along | 07:22 |
| ebowden | ...Why? | 07:23 |
| CaptHindsight | so it's all there, it just needs to be rewritten clearly and in order | 07:23 |
| ebowden | It's as if it's written in crayon. | 07:23 |
| CaptHindsight | yeah | 07:23 |
| CaptHindsight | like the POSaM doc, like you're sharing the info but not making it easy to follow | 07:24 |
| CaptHindsight | maybe they had several writers and just tried to hurriedly piece it all together at the last minute to meet someone deadline | 07:25 |
| CaptHindsight | that's how docs end up like this without an editor | 07:25 |
| kanzure | well they had no copyediting because they call it cyctosine a few times (instead of cytosine) | 07:25 |
| CaptHindsight | or try to write docs over IRC :) | 07:25 |
| CaptHindsight | no technical editor | 07:26 |
| CaptHindsight | How to disarm a bomb. 1) cut red wire 2) but not before cutting blue wire :) | 07:28 |
| chris_99 | haha | 07:30 |
| CaptHindsight | the ABI 391 uses glass beads and the inkjet uses glass slides | 07:31 |
| CaptHindsight | both use silane bonds to the fist base | 07:32 |
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| CaptHindsight | fist/first | 07:34 |
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| CaptHindsight | the ABI 391 floods the oligo building volume with each chem step by step but only build one type of oligo at a time | 07:37 |
| CaptHindsight | the inkjet can build 1M different oligos at a time but the ones built around the outer edges of each drop might have more errors | 07:38 |
| kanzure | probably some square of the distance dropoff law | 07:40 |
| CaptHindsight | so the washing, drying, rinsing steps don't wash away the baby oligos, they are bonded to the slide | 07:40 |
| kanzure | yup | 07:41 |
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| CaptHindsight | POSaM got lucky by discovering that Rain-X bonds to the glass slide, modifies the surface tension yet leaves space for the first base to be anchored to the slide | 07:44 |
| CaptHindsight | then again Rain-X isn't rocket science, just low cost and readily available, like finding out that reagents are available from the paint on Chinese toys or similar | 07:45 |
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| CaptHindsight | it would be nice to have photocleavable bond to the slide | 07:57 |
| CaptHindsight | just use a laser to selectively cleave and launch the infant oligos from the slides | 07:58 |
| CaptHindsight | in the order you want | 07:58 |
| CaptHindsight | but since you're printing them you can have in the order you wish anyway | 07:58 |
| CaptHindsight | I wonder if you could steer the launching electrostatically? | 08:00 |
| CaptHindsight | have them jump from the slides to an electrostatic array for ligation | 08:01 |
| CaptHindsight | or would you even have to | 08:01 |
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| CaptHindsight | selectively launch in the order you want into a ligation machine | 08:02 |
| CaptHindsight | and if ligation is done in parallel then you can have multiple launches and form longer stands more quickly | 08:03 |
| CaptHindsight | have to decide at what stages of this to have QC | 08:04 |
| CaptHindsight | you could also have beads on the tray and use an inkjet | 08:09 |
| CaptHindsight | bonds the beads to the tray and then cleave them when needed | 08:09 |
| CaptHindsight | so maybe inkjet + beads + laser launcher is as faster way to build oligos | 08:10 |
| CaptHindsight | as/a | 08:10 |
| CaptHindsight | between patents on inkjet and laser launched beads I can see why this has taken so long | 08:11 |
| CaptHindsight | kanzure: so what's the next step? | 08:13 |
| CaptHindsight | does anyone have access to one of the Cambrian bead launchers? | 08:14 |
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| kanzure | cambrian only has one or two as far as i know | 08:20 |
| CaptHindsight | ah I assumed that they were mass producing them | 08:21 |
| kanzure | CaptHindsight: next steps are things like "figure out the actual requirements for these reactions, and then make sure all requirements are satisfied" and "look at all the setup and shutdown procedures for posam, and figure out how much time and resources those require, and also document them better" | 08:21 |
| kanzure | no, they will never mass produce them-- they want to do in-house gene synthesis | 08:21 |
| CaptHindsight | so nobody will sell them for 15-20 years | 08:22 |
| CaptHindsight | they will all build them in house | 08:23 |
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| CaptHindsight | kanzure: let me know when you're ready to move forward | 08:25 |
| ParahSailin_ | the cambrian stuff doesnt work though | 08:29 |
| CaptHindsight | ParahSailin_: I thought it did | 08:30 |
| CaptHindsight | what does it actually do and not do? | 08:30 |
| ParahSailin_ | https://reason.com/blog/2015/06/30/rip-austen-heinz | 08:30 |
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| CaptHindsight | he could have asked for help in more ways than one | 08:45 |
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| CaptHindsight | Cambrian is up for sale before liquidation | 08:52 |
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| CaptHindsight | someone in linuxcnc is in charge of liquidating or finding buyers for Cambrian | 08:57 |
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| CaptHindsight | ok, so I'm right about my steps and how to QC using PCR and then launch only the good beads for ligation | 09:03 |
| CaptHindsight | for $4M you can have the whole Cambrian Lab | 09:06 |
| ParahSailin_ | hm, maybe ill tell affy to buy it | 09:07 |
| ParahSailin_ | is it in mtv? | 09:07 |
| ParahSailin_ | ah, sf | 09:08 |
| CaptHindsight | yeah, let me know if they want to meet the broker | 09:09 |
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| ParahSailin_ | oh yeah, do they have any swag? | 09:09 |
| ParahSailin_ | can i view the lab? | 09:10 |
| ParahSailin_ | collecting swag from these things is kinda my hobby | 09:10 |
| CaptHindsight | heh, they will check you bag at the door :) | 09:11 |
| ParahSailin_ | oh hey, theyre right by dropbox | 09:12 |
| ParahSailin_ | oh, fridge magents and pens fit in my pocket | 09:12 |
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| CaptHindsight | looks like they had one complete system and another partially assembled | 09:20 |
| CaptHindsight | the real trick was QC and using the laser to only launch know good beads | 09:21 |
| CaptHindsight | know/known | 09:22 |
| CaptHindsight | bbl after hands wake up | 09:22 |
| kanzure | 09:03 < CaptHindsight> ok, so I'm right about my steps and how to QC using PCR and then launch only the good beads for ligation | 09:26 |
| kanzure | was this from #linuxcnc? | 09:26 |
| kanzure | also, i wonder what anselm is going to be doing if he's not going to continue with cambrian genomics | 09:26 |
| CaptHindsight | it's a mess, looks like people are spooked by the death | 09:27 |
| CaptHindsight | how do people with so much $$ get so stupidstitious | 09:28 |
| CaptHindsight | somebody will probably buy the ip and sit on it | 09:29 |
| CaptHindsight | the lab will get sold for scrap/auction | 09:29 |
| CaptHindsight | 20 years later the next set of makers with reprap it | 09:30 |
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| CaptHindsight | still arguing over ramps vs 27 vs poopieboard v13 | 09:31 |
| CaptHindsight | for the controller | 09:31 |
| kanzure | cambrian genomics lab https://www.dropbox.com/sh/chg9tw7o2rv3ub1/AAAXeKmYUz4h7ERlPvd0SYUJa?dl=1 | 09:33 |
| kanzure | https://www.dropbox.com/sh/chg9tw7o2rv3ub1/AAAXeKmYUz4h7ERlPvd0SYUJa?dl=0 | 09:33 |
| kanzure | cambrian genomics assembly rig https://www.dropbox.com/sc/f5dupaq9bxn0jad/AADqcynzb0moLhlQv3XNxoIRa | 09:35 |
| ParahSailin_ | is that an illumina? | 09:39 |
| ParahSailin_ | oh, labcyte | 09:40 |
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| kanzure | i didn't realize that t12 was employed by cambrian genomics this whole time | 09:51 |
| kanzure | v. annoying | 09:51 |
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| kanzure | 09:42 < t12> honestly we just paid customarray to run their own machines to supply us with material | 09:59 |
| kanzure | 09:42 < t12> and we had one on site as a business partnership commitment and as a show peice | 09:59 |
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| kanzure | CaptHindsight: the main way to do gene assembly is by pipetting different dna fragments together into the same pot, then running a reaction. this needs to be done in parallel though for any reasonable scale to be reached. | 10:06 |
| CaptHindsight | yeah, one way | 10:07 |
| kanzure | well, tell me another way that works | 10:07 |
| CaptHindsight | i think we need to build a ligation machine | 10:07 |
| kanzure | you need to keep the beads separated (or tagged or visually tracked or something). you need to keep droplets separated. the ligation reactions don't yet work with >10 dna fragments in a reliable way. | 10:07 |
| CaptHindsight | cambrian took known good beads and then combined them | 10:08 |
| CaptHindsight | lets see what they used | 10:08 |
| kanzure | they used thermocyclers | 10:08 |
| kanzure | that's what the 20 machines in a line is | 10:08 |
| CaptHindsight | yuk | 10:08 |
| CaptHindsight | i was thinking of building small ones | 10:09 |
| kanzure | each of those cost $5k at least (thermocyclers are notoriously expensive for no good reason- it's really a <$200 machine in parts) | 10:09 |
| kanzure | my favorite pcr method is the one where you shoot an infrared laser beam at different droplets in an oil emulsion | 10:09 |
| kanzure | then you heat the droplets and cycle the temperature at each drop | 10:09 |
| CaptHindsight | yeah well, welcome to the world of scientific supply | 10:09 |
| kanzure | so the most basic design that i can think of that would work is having an array of pipettes that transport individual samples to a line of reaction chambers inside the same machine | 10:12 |
| CaptHindsight | ligation is done inside cells | 10:13 |
| kanzure | no | 10:13 |
| kanzure | i mean, let's not | 10:13 |
| CaptHindsight | we should improve on that design | 10:13 |
| CaptHindsight | down the road | 10:13 |
| kanzure | ok but that's like.. research. that's not going to work immediately. | 10:13 |
| fenn | t12> cambrian genomics | 10:14 |
| fenn | t12> the tech works | 10:14 |
| fenn | t12> it works well | 10:14 |
| fenn | t12> i build most of the 2nd gen of it | 10:14 |
| fenn | t12> but after ceo suicide everyone pretty much lost morale | 10:15 |
| fenn | for the record | 10:15 |
| kanzure | 08:53 < t12> including investors | 10:15 |
| kanzure | 08:54 < t12> i have weeks to round up a buyout pruced essentially on liquidation value | 10:15 |
| kanzure | 08:54 < t12> before it goes to asset liquidator | 10:15 |
| kanzure | 08:57 < CaptHindsight> t12: how many "laser printers" are there? | 10:15 |
| kanzure | 08:57 < t12> 3 depending on how you count. 1 that is reallyy rigged up | 10:15 |
| kanzure | 08:57 < t12> if yoy want to check it out sometime let me know | 10:15 |
| kanzure | 08:58 < t12> its a neat lab to check out | 10:15 |
| kanzure | 08:59 < t12> thats what cambrian really did | 10:16 |
| kanzure | 09:00 < t12> microarray purificatiin | 10:16 |
| kanzure | 09:00 < t12> its a giant in vitro cloning system | 10:16 |
| kanzure | 09:00 < t12> despite all the othrr statenents if what it is | 10:16 |
| kanzure | 09:01 < t12> make dna, critical dilution emulsion pcr to isolatr strands | 10:16 |
| kanzure | 09:02 < t12> barcode the strands | 10:16 |
| kanzure | 09:02 < t12> sewuence them in illumina, and our custom sequencer | 10:16 |
| kanzure | 09:02 < t12> that gives yoy map of immobalized beads to correct beads | 10:16 |
| kanzure | 09:02 < t12> eject those | 10:16 |
| kanzure | 09:07 < t12> competitor beat us to the core product, maybe | 10:16 |
| kanzure | 09:08 < t12> due to better management really | 10:17 |
| kanzure | a line of 20 thermocyclers should not be the design goal here | 10:23 |
| kanzure | that's just... poor planning. | 10:23 |
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| kanzure | https://patents.google.com/patent/US20140309119A1/en?assignee=Gen9 | 11:23 |
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| kanzure | 10:54 < t12> re lineup of pcr machines, pcr in bulk is suprisingly annoying | 12:19 |
| kanzure | 10:54 < t12> https://www.dropbox.com/s/ydjowb969ak3ka0/20150601-20150601-_6010050.jpg?dl=0 that thing can do like 200 plates at a time though | 12:19 |
| kanzure | 10:55 < t12> all the customarray r&d was in their electrode, not the chemistry | 12:19 |
| kanzure | 11:36 < t12> the real guts for us lie in emulsion pcr techniques | 12:22 |
| kanzure | 11:36 < t12> which are a nightmare to develop | 12:22 |
| kanzure | 11:37 < t12> so theres all kinds of expensive/slow cleanups you can do | 12:22 |
| kanzure | 11:37 < t12> like you can gel purify what you've made on the beads to select for some length | 12:22 |
| kanzure | 11:38 < t12> nanopores are just finnecky as hell | 12:22 |
| kanzure | 11:38 < t12> and very high error rates | 12:22 |
| kanzure | 11:52 < t12> i'd say the hangups are first how to actually design the machine, second is successfully building them | 12:24 |
| kanzure | 11:53 < t12> like in the protein world taking a structure and rationally making it be better at some thing is very hard | 12:24 |
| kanzure | 11:53 < t12> and kinda laughed at | 12:25 |
| kanzure | 11:53 < t12> how it seems to really be done is maybe some human guidance, and alot of shotgun expirements and screening | 12:25 |
| kanzure | 11:53 < t12> mutagenesis expirements cover the problem space faster than human mind rationality | 12:25 |
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| ParahSailin_ | kanzure: hear that? customarray | 12:41 |
| ParahSailin_ | surprised the tech "works well" | 12:43 |
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| CaptHindsight | he just had other issues | 12:43 |
| ParahSailin_ | if tech works well, then just do what they are doing, not this whole inkjet shit | 12:43 |
| CaptHindsight | the tech worked | 12:43 |
| ParahSailin_ | no need to waste time when theres already proven way to do it | 12:45 |
| CaptHindsight | it wasn't optimal, but it worked | 12:46 |
| CaptHindsight | I mean the setup wasn't optimal | 12:46 |
| CaptHindsight | it evolved irrationally | 12:46 |
| ParahSailin_ | works or it didnt? | 12:46 |
| CaptHindsight | it still works | 12:47 |
| CaptHindsight | the assembly like just evolved irrationally | 12:47 |
| CaptHindsight | like/line | 12:47 |
| ParahSailin_ | so then build it correctly? | 12:47 |
| CaptHindsight | so nobody quit your days jobs yet | 12:48 |
| CaptHindsight | well that requires a plan and funding | 12:48 |
| ParahSailin_ | isnt that what you guys are doing? | 12:49 |
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| CaptHindsight | http://www.ebay.com/itm//201388723543 | 12:53 |
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| fenn | it looked expensive | 13:07 |
| fenn | $4 million to be precise | 13:07 |
| fenn | that was a response to ParahSailin_ | 13:09 |
| CaptHindsight | yeah t12 said $4m | 13:15 |
| CaptHindsight | or it goes to auction is 2 weeks | 13:15 |
| CaptHindsight | ebay for parts in 3 :) | 13:15 |
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| kanzure | i never said that cutomarray didn't work | 13:51 |
| kanzure | $4M was the liquidation potential price i think, not the actual cost of building it | 13:53 |
| kanzure | may be more or less | 13:53 |
| CaptHindsight | should have been far less but then again who knows | 13:55 |
| kanzure | i was talking with fenn earlier today, | 13:57 |
| kanzure | i proposed wax printing and wax melting | 13:57 |
| kanzure | to separate drops | 13:57 |
| kanzure | you could use infrared or heated pin needles | 13:58 |
| kanzure | you could also have different wax types that melt at different temperatures | 13:58 |
| kanzure | that sounds like a thing | 13:58 |
| kanzure | i wonder if the wax would contaminate the chemistry though | 13:58 |
| kanzure | (er, more specifically, the wax heating would be to make the drops mix and touch somehow) | 14:00 |
| kanzure | oh right, if you heat wax then it will just melt and mix with the other chemicals. that's not good. | 14:00 |
| fenn | the idea here being that the well walls are made of wax and when you melt one wall with a laser the two droplets combine into one droplet | 14:04 |
| fenn | i also suggested inkjetting a line of soap between droplets to break down the hydrophobic barrier | 14:05 |
| fenn | the point being you combine droplets on the plate without doing a bunch of pipetting | 14:05 |
| fenn | now that i think about it though, soap would reduce the surface tension for the whole droplet and it would go everywhere | 14:06 |
| CaptHindsight | whats wrong with electrostatic guides? | 14:08 |
| fenn | what is that? | 14:08 |
| fenn | like a UV laser generating a surface charge? | 14:08 |
| CaptHindsight | kanzure: an inert wax | 14:08 |
| kanzure | are there inert waxes? | 14:09 |
| fenn | most waxes are relatively inert already | 14:09 |
| CaptHindsight | inert from the DNA perspective | 14:09 |
| CaptHindsight | or we can make a low Tg oligomer | 14:10 |
| CaptHindsight | reflow it by heating with a laser | 14:10 |
| kanzure | it's not just dna inertness that matters, but also all the other chemicals in the system- unless the wax is never touched by any of them | 14:11 |
| kanzure | not sure what a tg oligomer is | 14:11 |
| CaptHindsight | what problem are you trying to solve? | 14:12 |
| kanzure | genome synthesis | 14:12 |
| CaptHindsight | kanzure: we can tailor the Tg of the oligomer | 14:12 |
| CaptHindsight | heh, what step? | 14:12 |
| fenn | if there are a million spots you have to combine a small number of them at a time or the oligos will not be able to find their one true love | 14:13 |
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| CaptHindsight | you can plan the order/ their arrangement | 14:13 |
| CaptHindsight | https://www.youtube.com/watch?v=u2MK81wWQPM what do they call these for fluids? | 14:14 |
| fenn | programmable droplet array | 14:15 |
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| kanzure | "EOWD" stuff | 14:15 |
| kanzure | .title | 14:15 |
| yoleaux | Thermal-Biomorph/Electrostatic Organic Ciliary Array (the array manipulates parts) - YouTube | 14:15 |
| CaptHindsight | the hurdle seems to be QC like t12 mentioned | 14:15 |
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| kanzure | er, he only said that because he was using a pre-made dna synthesis system | 14:16 |
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| CaptHindsight | the rest is academic | 14:16 |
| kanzure | CaptHindsight: new plan is that we'll move forward, but we're shipping everything to fenn instead | 14:17 |
| CaptHindsight | I'll look into ligation on the nanoscale | 14:17 |
| CaptHindsight | a small machine that does QC and makes and or repairs connections | 14:18 |
| fenn | aka ligase | 14:18 |
| CaptHindsight | that would appear to be ideal | 14:18 |
| CaptHindsight | a nano-ligator | 14:18 |
| fenn | .wik ligase | 14:18 |
| yoleaux | "In biochemistry, ligase (from the Latin verb ligāre — "to bind" or "to glue together") is an enzyme that can catalyze the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of a small pendant chemical group on one of the larger molecules or the enzyme catalyzing the linking together of two …" — https://en.wikipedia.org/wiki/Ligase | 14:18 |
| kanzure | wax printing would be inconvenient on this system, because you need to print wax at a different size (in between all the other spots) | 14:19 |
| kanzure | and it needs to be continuous | 14:19 |
| kanzure | or something | 14:19 |
| CaptHindsight | yeah, just didn't use the term | 14:19 |
| CaptHindsight | kanzure: you make a template and mass produce them | 14:19 |
| kanzure | perhaps, but only if you can keep them clean | 14:19 |
| CaptHindsight | wax coated slides | 14:19 |
| kanzure | you could also mill a multi-layer wax surface | 14:20 |
| CaptHindsight | with the proper space and channels | 14:20 |
| kanzure | (or laser heat it anyway) | 14:20 |
| kanzure | if you heat it, where does the wax go.. it will just pool and block everything once it cools. | 14:20 |
| fenn | polyimides (kapton) have the convenient property of sublimating instead of melting | 14:21 |
| kanzure | perhaps if you can . | 14:21 |
| kanzure | ah | 14:21 |
| juri_ | um. | 14:21 |
| fenn | probably not very inert tho | 14:22 |
| kanzure | what about just giant wax walls that don't change. the wax wall surrounds 10 spots. then you add liquid into that container to mix them. | 14:23 |
| juri_ | call me stupid, but, why not use a wax containing magnetic particles? heat the wax with a laser, and let a magnet under the slide pull the half melted wax to the bottom, allowing your droplets to mingle at the surface? | 14:23 |
| kanzure | so it is an elevated surface, that you can flood at your convenience | 14:23 |
| CaptHindsight | if you inkjet onto a programmable droplet array you can move things around, but how about QC? | 14:23 |
| kanzure | droplet arrays have their own host of problems... electrowetting etc. is something that jonathan cline experimented with, but his results- while interesting- do not seem practical for this sort of project. | 14:24 |
| fenn | a wax with higher density than water would accomplish the same thing | 14:24 |
| fenn | actually, are these droplets even made of water? | 14:24 |
| kanzure | i think that an elevated wax surface (not requiring melting or sublimation) would work. | 14:24 |
| kanzure | i don't know what the droplets are made of | 14:24 |
| CaptHindsight | at what point are you speaking? | 14:25 |
| kanzure | at any step | 14:25 |
| juri_ | fenn: fair point. | 14:25 |
| kanzure | at all steps | 14:25 |
| kanzure | for pcr and ligation it's water but that's all i know | 14:25 |
| kanzure | elevated wall approach (for different "flood groups") allows for any material, not just wax- could be metal, teflon, whatever. | 14:26 |
| CaptHindsight | what is in the wells? | 14:27 |
| kanzure | same thing as always | 14:27 |
| CaptHindsight | beads, free floating oligos? | 14:27 |
| kanzure | "flood groups" are for mixing things together | 14:27 |
| kanzure | "mix groups" | 14:27 |
| kanzure | the point is that if you flood it with a liquid that you know which spots are going to be covered in the liquid, and it's some subset of the total set of spots | 14:28 |
| kanzure | (inverted pyramid) | 14:29 |
| kanzure | whoops i mean flipped pyramid. i'm not sure what an inverted pyramid would be. | 14:29 |
| CaptHindsight | potato potato | 14:30 |
| CaptHindsight | storms are over bbl | 14:30 |
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| kanzure | man i hate gimp | 14:49 |
| kanzure | here is an illustration of the "flood group" concept http://diyhpl.us/~bryan/papers2/DNA/flood-groups.png | 14:50 |
| kanzure | however, the larger "flood groups" have more volume and thus require more fluid- and the concentration of the oligos is going to be lower.... | 14:50 |
| kanzure | also i think there would be 10 members of each group | 14:52 |
| kanzure | so there would be 9 height-depths if the plan is to have 1 million spots | 14:53 |
| kanzure | oh, i guess 10. it depends on whether height=0 counts as a height. | 14:53 |
| kanzure | if there is an inert sublimating compatible wax, then that might be preferable because you would presumably have some way of using much less liquid by volume for your later reactions (and thus higher oligo concentrations) | 14:56 |
| kanzure | also perhaps it's possible to use gas to push droplets around and merge with other droplets. if that's the case then a lot of this is pointless. | 14:58 |
| kanzure | (you could use a syringe pump plus needle to blow nitrogen or argon at droplets) | 14:59 |
| kanzure | .title https://www.youtube.com/watch?v=YphnDXf4Vm4 | 15:04 |
| yoleaux | Single cell membrane poration by bubble-induced microjets in a microfluidic chip - YouTube | 15:04 |
| kanzure | hmm that's not what i wanted | 15:04 |
| kanzure | .title https://www.youtube.com/watch?v=Mw_MaFA9mhc | 15:07 |
| yoleaux | Droplets falling from a pipette - YouTube | 15:07 |
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| streety | the idea being the oligos in group 1 and 2 combine in the first flood fill, then the pool from groups 1 and 2 combine in the second flood fill, and so on? | 15:10 |
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| kanzure | streety: correct | 15:13 |
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| streety | would there be any issues in spreading of the dot/spots with the increased separation between substrate and print head? | 15:16 |
| fenn | probably not | 15:17 |
| kanzure | printhead would be done, or just depositing enzymes and extra reagents anywhere- and lots of 'em | 15:17 |
| kanzure | perhaps you could use a syringe pump + needle to blast nitrogen into each flooded area to mix it (but not enough to blow all the junk out) | 15:20 |
| streety | I came across http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00062 recently, at least somewhat relevant | 15:20 |
| kanzure | .title | 15:20 |
| yoleaux | An Error Occurred Setting Your User Cookie | 15:20 |
| kanzure | "A Versatile Microfluidic Device for Automating Synthetic Biology" (keasling) | 15:21 |
| kanzure | or maybe this is a different keasling | 15:21 |
| kanzure | this looks like it's doing assembly only (which is fine, but just a caveat) | 15:22 |
| kanzure | also claims on-chip electroporation and incubation | 15:22 |
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| kanzure | .g russian pet foxes | 16:22 |
| yoleaux | http://www.youtube.com/watch?v=d1G2yZMUNUQ | 16:22 |
| kanzure | .title | 16:22 |
| yoleaux | Russian Domesticated Foxes - YouTube | 16:22 |
| kanzure | .g russian pet foxes -site:youtube.com | 16:22 |
| yoleaux | http://www.fastcompany.com/3037451/pet-week/meet-your-new-pet-a-domesticated-fox | 16:22 |
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| kanzure | you could also use hydrophobic blunt ends to push around droplets i think | 17:47 |
| kanzure | .title https://www.youtube.com/watch?v=MkLbVLGcn-A | 17:48 |
| yoleaux | Superhydrophobic Water - Part II - YouTube | 17:48 |
| kanzure | .title https://www.youtube.com/watch?v=qHrBhgwq__Q | 17:49 |
| yoleaux | Science off the Sphere: Knitting Needle Experiment - YouTube | 17:49 |
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| kanzure | .title https://www.youtube.com/watch?v=RM6m7GcSKx8 | 17:51 |
| yoleaux | Cutting a water droplet using a superhydrophobic knife - YouTube | 17:51 |
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| kanzure | ah here we go, this is what i want: | 17:53 |
| kanzure | .title https://www.youtube.com/watch?v=UhwU2VDyrH4 | 17:53 |
| yoleaux | Drag water droplet - YouTube | 17:53 |
| kanzure | i guess sometimes it doesn't work...? https://www.youtube.com/watch?v=EWnx1-iChbA | 17:58 |
| kanzure | there's also the marangoni flow method but i don't know how to hook it up to a non-microfluidic dna synthesizer | 18:05 |
| kanzure | .title https://www.youtube.com/watch?v=DRR6-qgHB-I | 18:06 |
| yoleaux | Superhydrophobic teflon surface - YouTube | 18:06 |
| kanzure | "I took teflon/PTFE and roughened it with 240 grit wet and dry paper. It is very cool to watch but the surface is delicate, rub it with your finger and you bruise the structure (I assume) and it stops working." | 18:07 |
| kanzure | dat one. | 18:09 |
| kanzure | he has a bunch of machine shop videos | 18:09 |
| kanzure | also he did a printer https://www.youtube.com/watch?v=a14zELKPw8M | 18:11 |
| kanzure | .wik fluorinert | 18:12 |
| yoleaux | "Fluorinert is the trademarked brand name for the line of electronics coolant liquids sold commercially by 3M. It is an electrically insulating, stable fluorocarbon-based fluid which is used in various cooling applications. It is mainly used for cooling electronics." — https://en.wikipedia.org/wiki/Fluorinert | 18:12 |
| CaptHindsight | HP gen1 tij looks like | 18:14 |
| CaptHindsight | https://www.youtube.com/watch?v=iSZc5I90ddA Epson head shown here | 18:17 |
| CaptHindsight | https://www.youtube.com/watch?v=pLbHvBvChSk another Epson, turn down the reggae mon | 18:18 |
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| kanzure | so flooding vs. pushing drops around? | 19:39 |
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| ParahSailin_ | cant believe that instead of going to jvm, erlang etc to make concurrency work, dropbox hired guido instead to make their python go | 20:26 |
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| drethelin | http://lesswrong.com/r/discussion/lw/mht/i_have_just_donated_10000_to_the_immortality_bus/#comments | 22:10 |
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| kanzure | "... ... this is a bad reality show for personal promotion, nothing more and nothing less. It's not even a good one. I just showed the indiegogo page to a friend who literally called it 'facepunch worthy'." | 23:01 |
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| delinquentme | kanzure, i didnt pay attention in #linuxcnc | 23:08 |
| delinquentme | what was the TLDR? | 23:09 |
| kanzure | ctrl-f t12 http://gnusha.org/logs/2015-07-18.log | 23:12 |
| kanzure | emulsion pcr stuff https://gtc.soe.ucsc.edu/content/emulsion-pcr-and-bead-enrichment | 23:14 |
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| kanzure | "single-molecule emulsion pcr in microfluidic droplets" http://link.springer.com/article/10.1007%2Fs00216-012-5914-x | 23:15 |
| kanzure | emulsion pcr infographic.. thing.. http://users.ugent.be/~avierstr/nextgen/emulsionpcr.jpg | 23:15 |
| kanzure | hmm that seems oddly specific. i would have guessed many beads per drop. | 23:16 |
| kanzure | oh they probably mean one-library-member-specific bead, not one bead.. | 23:17 |
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| --- Log closed Sun Jul 19 00:00:15 2015 | ||
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