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| archels_ | kanzure: G-protein coupled optogenetics sounds relatively slow. Did they combine it with the usual opsins or as a replacement thereof? | 03:05 |
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| archels_ | woah, even slower that I thought. Timescale of seconds. | 03:06 |
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| kanzure | .title http://www.c2o.pro.br/en/automation/x73.html | 07:16 |
| yoleaux | Building a Respirometer using FSM, Tcl and Arduino | 07:16 |
| kanzure | "lab devices with rs232 interfaces" http://www.c2o.pro.br/en/automation/x45.html | 07:17 |
| kanzure | indiebio: hi | 07:17 |
| kanzure | .title https://news.ycombinator.com/item?id=9894570 | 07:22 |
| yoleaux | 1.5 TB of Dark Net Market scrapes | Hacker News | 07:22 |
| indiebio | hi kanzure | 07:39 |
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| kanzure | stopping a comet http://web.archive.org/web/20140415063820/http://szabo.best.vwh.net/comet.thread.html | 07:52 |
| kanzure | self-replication stuff http://szabo.best.vwh.net/nano.musings.html | 07:53 |
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| kanzure | curious approach to keeping track of stack depth for context switching https://news.ycombinator.com/item?id=9896462 | 09:12 |
| kanzure | http://faq.sealedabstract.com/uninterruptible_programming_supply/ | 09:13 |
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| kanzure | long conference talk on adhd https://www.youtube.com/watch?v=SCAGc-rkIfo | 09:18 |
| nmz787_i | unfortunate problem for ppl suffering, as they can't pay attention long enough | 09:22 |
| nmz787_i | (to get through it) | 09:22 |
| nmz787_i | .title https://www.youtube.com/watch?v=04aIN3T7whg | 09:22 |
| yoleaux | JAMES BROWN Make It Funky cd set - YouTube | 09:22 |
| kanzure | working memory defecits https://www.youtube.com/watch?v=SCAGc-rkIfo&t=19m40s | 09:28 |
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| nmz787_i | kanzure: any ideas for bonding a top-plate to a nano-channel between 2 microchannels? | 09:42 |
| kanzure | does the bonding problem change because of the channels...? | 09:43 |
| nmz787_i | something that would look like this http://imgur.com/sXY1N8P | 09:47 |
| nmz787_i | I guess the concern is that the scale on the small channel is so small, any imperfection in bonding will be a big deal | 09:47 |
| nmz787_i | if you tried to use glue, it might block the channel, or not compress fully and make the channel effectively larger | 09:48 |
| nmz787_i | or the plate might not be flat enough | 09:48 |
| nmz787_i | I'm not sure what the equivalent of a wafer-polish glass cover-slip would be | 09:48 |
| nmz787_i | I guess I could get some cover slip glass characterized with an interferometer | 09:49 |
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| nmz787_i | or maybe at that scale the FIB/SEM would be fine | 09:50 |
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| nmz787_i | hmm http://www.ebay.com/itm/GATAN-PRECISION-ION-MILLING-MACHINE-WITH-CHAMBER-/271212366622?pt=LH_DefaultDomain_0&hash=item3f25844b1e | 09:56 |
| nmz787_i | $1000 | 09:56 |
| nmz787_i | I'm not sure what the spot size is, and if its seeming manual-ness could be amended with newage tech | 09:56 |
| nmz787_i | maybe 40nm? | 09:57 |
| nmz787_i | https://hal.archives-ouvertes.fr/jpa-00229994/document | 09:57 |
| nmz787_i | "The ion beam diameter is dependent on the voltage used and the objective and condenser lens excitations. The smallest beam diameter that can be obtained is approximately 1.5 pm (FWHM) at 10 kV. T" | 09:58 |
| nmz787_i | not sure of the model | 09:58 |
| nmz787_i | "A commercial Gatan model 645 precision ion milling system was used for this investigation. Selected portions of the specimen can be ion milled at a chosen voltage (1, 2, 4, 6, 8, or 10 kV) with a rastered ion beam generated from for example a noble gas, hydrogen, nitrogen or oxygen. Argon is the most commonly used ion beam source. The ion beam diameter is dependent on the voltage used and the objective and condenser lens excitations." | 09:59 |
| kanzure | adhd is time travel https://www.youtube.com/watch?v=SCAGc-rkIfo&t=1h12m50s | 10:01 |
| CaptHindsight | unless the chamber is beyond repair that ion beam is a good deal | 10:07 |
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| kanzure | i think they hired richard dreyfuss for this lecture | 10:22 |
| CaptHindsight | was a home found for the inkjet? | 10:25 |
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| kanzure | CaptHindsight: yep, los angeles biohackers | 10:28 |
| kanzure | or nmz787 | 10:28 |
| CaptHindsight | nmz787: are you in the LA area? | 10:29 |
| CaptHindsight | http://www.biohackers.la/ this group? | 10:30 |
| kanzure | yes that group | 10:39 |
| kanzure | nmz787 is in portland, oregon | 10:39 |
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| fenn | wiki.biohackers.la is down so it's hard to judge how active the group is | 10:42 |
| fenn | "Ignore the smell - it's not us." | 10:43 |
| CaptHindsight | http://www.meetup.com/LAbiohackers/ | 10:55 |
| kanzure | didn't we backup their wiki at some point | 10:56 |
| kanzure | was that a thing we did? | 10:56 |
| CaptHindsight | reading through the member list they have some actual biologists | 11:00 |
| nmz787_i | yeah and some good doctors | 11:01 |
| nmz787_i | phds | 11:01 |
| nmz787_i | at least one | 11:01 |
| nmz787_i | and a scrappy teenager | 11:01 |
| nmz787_i | scrappy may not be the right word | 11:01 |
| CaptHindsight | every team needs a mascot | 11:01 |
| heath | we're looking for a place to house a dna synthesizer still? | 11:02 |
| heath | i have a workshop in the backyard and a guest room for anyone who wants to visit | 11:02 |
| nmz787_i | seems not he | 11:02 |
| nmz787_i | heath^ | 11:02 |
| heath | just thought i'd toss that in the mix | 11:03 |
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| fenn | ok so it looks like la biohackers actually have events and people attend those events | 11:06 |
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| fenn | was there a summary of this "bioprinting meetup" anywhere? http://i.imgur.com/Xr5xdvg.png | 11:08 |
| kanzure | no i didn't see a summary | 11:08 |
| kanzure | they are not optimal on the internet presence axis | 11:08 |
| kanzure | but neither is counter culture labs | 11:08 |
| juri_ | that's a problem at BUGS, too. | 11:16 |
| fenn | it's really hard to tell if the reason for no internet presence is poor computer skills or just poor attendance | 11:18 |
| fenn | btw juri it's BUGSS | 11:18 |
| fenn | impossible to google without the extra S | 11:18 |
| kanzure | it can't be poor attendance- surely even a single person should be publishing shit on the web | 11:19 |
| kanzure | how hard is it to send out an email before/after each event, jeeze | 11:19 |
| kanzure | another option is that i could start renting some space somewhere, but i would want some of you jackasses to move to be close to the location | 11:20 |
| kanzure | and i can't just take the geographic average to pick a location, because that would put it in the middle of the ocean or something | 11:20 |
| juri_ | fenn: good point. thanks. ;) | 11:21 |
| fenn | kanzure: what happened with the lab in carlsbad? | 11:22 |
| kanzure | jojack is still doing that | 11:23 |
| kanzure | http://biotechnbeyond.com/ | 11:23 |
| fenn | is there a functioning community and people who know which end of the pipette to use? | 11:23 |
| kanzure | yashgaroth and jcline have apparently stopped attending regularly | 11:23 |
| kanzure | i believe he is just renting out benches | 11:24 |
| fenn | benches do not a lab make | 11:24 |
| kanzure | well, there's equipment of course | 11:25 |
| fenn | hmm @ http://htsresources.com/piezoelectric_dispensing_robotic_platforms.php | 11:31 |
| kanzure | http://diyhpl.us/~bryan/papers2/DNA/oligonucleotide-synthesis-by-phosphoramidite-method-cycle-diagram.png | 11:31 |
| fenn | .wa 60 picoliter sphere diameter | 11:34 |
| yoleaux | sphere: volume 60 pL (picoliters): diameter: 2 3^(2/3) (5/pi)^(1/3) pL^(1/3) (cube root picoliters)~~4.85718 pL^(1/3) (cube root picoliters); Visual representation: http://is.gd/PtXy3w; Unit conversions: 0.001912 inches; 48.57 µm (micrometers); 0.04857 mm (millimeters); Comparisons as diameter: ~0.6 × diameter of an average human hair (~80 µm); ~2 × wool fiber diameter (~20 µm); ~5 × typical cotton fiber diameter (~10 µm) | 11:34 |
| kanzure | 50 micron diameter, not bad | 11:36 |
| kanzure | maybe we should put it in a trailer and just buy a truck | 11:37 |
| fenn | you're missing the point | 11:37 |
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| fenn | in order to get the machine to a point where it reliably does something useful, a person in a lab has to work with the machine for a period of time | 11:38 |
| fenn | if you want to just stick it in a storage facility to gather dust, that's kinda pointless | 11:38 |
| kanzure | i wonder if azco biotech would tweak the machine for us, they said they offer design services | 11:38 |
| kanzure | machine design services | 11:39 |
| CaptHindsight | what would they tweak? | 11:39 |
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| CaptHindsight | what in the machine design needs tweaking? | 11:39 |
| fenn | lots of things need to be verified | 11:40 |
| kanzure | reaction conditions, yield, chemistry, reagents | 11:40 |
| kanzure | dilution | 11:40 |
| CaptHindsight | rinse and dry cycles need some common sense | 11:40 |
| kanzure | concentration | 11:40 |
| kanzure | binding beads to the surface, binding oligos to beads or to surfaces, releasing oligos from beads and/or surfaces | 11:40 |
| CaptHindsight | ahh, thats all chemistry not mechanics | 11:40 |
| fenn | once we've verified that it produces oligos, starting on assembly procedures and scaling up the number/length of fragments | 11:41 |
| fenn | and getting errors to a reasonable level | 11:41 |
| fenn | i'm not worried about mechanics really | 11:42 |
| kanzure | there's also additional problems that you may already be familiar with like clogging, purging, leaking, unwanted spot blending, .. | 11:42 |
| kanzure | mechanics are easy | 11:43 |
| kanzure | enclosure leaks... | 11:43 |
| kanzure | finding solvents compatible with the mechanical components | 11:43 |
| kanzure | (and plastic components) | 11:44 |
| fenn | finding solvents that aren't a huge pain in the ass to deal with after being used | 11:44 |
| CaptHindsight | kanzure: fluid compatibility with the heads will come with the experience of using it | 11:44 |
| CaptHindsight | you want to keep the fluids moving | 11:44 |
| kanzure | then there's even weirder problems like, "okay the chemical reaction should be happening, why isn't it decapping? is it decapping at all? is the reaction proceeding as expected? whatdo" | 11:44 |
| CaptHindsight | run cleaning cycles | 11:45 |
| kanzure | hah maybe we should just email them, sales@azcobiotech.com | 11:45 |
| CaptHindsight | did the POSaM users not publish their experience with that? | 11:46 |
| fenn | i'll take this opportunity to point out that POSAM was designed for creating hybridization arrays, not gene synthesis | 11:46 |
| fenn | they didn't have to worry about yield or error rate really | 11:46 |
| CaptHindsight | yeah, we've been around this circle before | 11:47 |
| kanzure | yield/error rate is different from "oshit i don't have any oligos whatsoever" | 11:48 |
| CaptHindsight | you have to be able to print the bases and the chems to links them, that is the first step | 11:49 |
| fenn | (btw the htsresources link was related somehow to biotechnbeyond) | 11:50 |
| CaptHindsight | then you learn how to link them into longer sections | 11:50 |
| kanzure | "first step".. except for all the other first steps (linking a precursor to the surface of the array or to the surface of a bead on the array) | 11:51 |
| fenn | the first step... installing a bunch of infrastructure so the machine can work | 11:51 |
| fenn | first you start with the universe | 11:52 |
| CaptHindsight | ok, done :) | 11:52 |
| kanzure | er, i think binding the oligos ot the surface is in-scope | 11:52 |
| kanzure | otherwise your argon blaster is going to blow your oligos away | 11:52 |
| CaptHindsight | argon blaster vs argon positive pressure | 11:53 |
| kanzure | oligos are just molecules floating around, unless you link them to the surface | 11:54 |
| CaptHindsight | they invented 3 different program languages vs use G-code | 11:54 |
| fenn | i dont get this page, it says "projects" but it's all pieces of equipment: http://biotechnbeyond.com/projects/ | 11:54 |
| CaptHindsight | if you force yourself to read their program they do list the times spent for the steps | 11:54 |
| kanzure | don't worry about the software | 11:55 |
| fenn | so, what's a typical linker for starting an oligo? | 11:55 |
| kanzure | https://en.wikipedia.org/wiki/Oligonucleotide_synthesis#Linker_chemistry | 11:56 |
| CaptHindsight | isn't it place a drop of base, then dry then place a drop, dry and then something to cause them to link, rinse, dry | 11:56 |
| juri_ | do we have wiki pages on this machine yet? | 11:57 |
| CaptHindsight | kanzure: not, just looking at the printing steps they used | 11:57 |
| kanzure | juri_: you have never edited the wiki | 11:57 |
| CaptHindsight | are those actual programs or just garbage examples? | 11:57 |
| kanzure | CaptHindsight: what do you mean by "dry". in your head, what do you see happening to the oligos when you "dry"? | 11:57 |
| juri_ | kanzure: indeed. | 11:57 |
| kanzure | those programs are worthless; ignore them. i have read the source code and i have extracted all that i need to know from them. | 11:57 |
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| CaptHindsight | kanzure: are they evaporating the vehicle/solvent that the bases are suspended in? | 11:58 |
| fenn | i think the "dry" step is after washing, to get rid of the macroscopic quantities of solvent | 11:58 |
| fenn | the drops evaporate on their own relatively quickly(?) | 11:59 |
| CaptHindsight | I though that you guys had this all figured out | 11:59 |
| kanzure | if you evaporate then you lose the oligos, unless the oligos are attached to something | 11:59 |
| fenn | the inkjet drops i mean | 11:59 |
| CaptHindsight | though/thought | 11:59 |
| kanzure | oh yes sorry that i don't have a complete design for a genome compiler, i'll get right on that after i get back from fighting crime as batman | 11:59 |
| fenn | kanzure: huh? evaporation means the solvent goes away, not the oligos | 12:00 |
| kanzure | fenn: if the oligos are not linked to anything, they will be in the evaporate | 12:00 |
| kanzure | i think | 12:00 |
| fenn | wrong | 12:00 |
| CaptHindsight | do they link the bases while they are still in the vehicle/solvent (liquid that suspends them)? | 12:00 |
| kanzure | CaptHindsight: http://diyhpl.us/~bryan/papers2/DNA/oligonucleotide-synthesis-by-phosphoramidite-method-cycle-diagram.png | 12:00 |
| fenn | in this chemistry, the bases link to either a linker that is covalently bound to a solid surface, or a growing strand of bases attached to the linker | 12:01 |
| fenn | the linker is not shown in that diagram | 12:01 |
| kanzure | evaporating away the solvent is news to me. i thought it was just argon blowing away everything not surface-attached. | 12:02 |
| fenn | also i think in step 3 it should be "single-base insertions" not deletions | 12:02 |
| CaptHindsight | ok, then which is the preferred method or should we support both? | 12:02 |
| kanzure | method details are currently not completely know | 12:02 |
| kanzure | *known | 12:02 |
| kanzure | although i may speak with authority, i technically don't know all of the necessary reaction conditions | 12:02 |
| fenn | CaptHindsight: it's the same method, not two methods | 12:02 |
| fenn | you start with just a linker, then extend on top of the first base, then extend on top of the second base, etc | 12:03 |
| kanzure | evaporation is only mentioned twice in that wikipedia article, and it's not about solvents | 12:03 |
| CaptHindsight | should we covalently bond the first base to something on the sample tray? | 12:03 |
| fenn | yes | 12:04 |
| CaptHindsight | ok, solved. next question :) | 12:04 |
| fenn | how do we get the spot size of the first base/linker to be smaller than the rest of the spots | 12:04 |
| kanzure | er i would consider it solved once you know which linker to use, how to make the linkers, how to cleave the linkers | 12:05 |
| kanzure | fenn: is that a problem? why is that necessary? | 12:05 |
| kanzure | oops and also how to apply the linkers to the surface | 12:05 |
| fenn | the first spot has to be smaller or else you get weird incomplete sequences at the edges where they partially overlap | 12:05 |
| kanzure | i think beads only work with the microwell approach because otherwise the bead is bigger than the spot on the surface | 12:06 |
| fenn | the bead is bigger than 10 microns? | 12:06 |
| kanzure | but if you were using a trench/pore/well, then presumably you could guarantee mixing inside of that | 12:06 |
| kanzure | well assume a 10 micron diameter, is the droplet on the surface going to have that height at least? | 12:06 |
| kanzure | well anyway; this seems like a quality control issue that could be solved later | 12:07 |
| kanzure | whereas linker chemistry is not quality control | 12:07 |
| kanzure | we need a chemist | 12:10 |
| CaptHindsight | need a linker? | 12:11 |
| kanzure | need an oligo-compatible linker that doesn't impact the rest of the reaction | 12:12 |
| kanzure | so probably just steal one from one of the papers | 12:12 |
| kanzure | or azco might sell something appropriate | 12:12 |
| CaptHindsight | I though that this was all well understood and thought through already | 12:12 |
| fenn | kanzure: the linker should be shielded by the base so it shouldn't really matter once the first base is attached | 12:14 |
| fenn | you want to use up all of the linkers in the first reaction anyway | 12:14 |
| CaptHindsight | I have time to build, assemble and scrounge for deals on parts, not much for the chemistry since it requires so much time to process and has a short shelf life | 12:15 |
| fenn | i was thinking the first few bases would be throwaway like AAA and then a restriction enzyme sequence for cleaving | 12:17 |
| fenn | it would be best to not have any sequence restrictions though (pun not intended) | 12:17 |
| kanzure | what we need is a document that clearly identifies all of the necessary reaction conditions for every step, which is something that i haven't assembled yet | 12:19 |
| kanzure | i imagine that it would be based on a bunch of "supplementary material" documents | 12:19 |
| kanzure | and also, we should have at least one document where we show the math on concentrations or the parameters for the chemical reactions, including electron flow | 12:20 |
| fenn | electron flow? | 12:20 |
| fenn | there are no redox reactions afaik | 12:20 |
| kanzure | i mean electron availability | 12:23 |
| kanzure | ". Even a modest deceleration of Moore's law can have a dramatic effect: a five per cent reduction in the pace of gate-length shrinkage -- from 16 percent to 11 per cent per year -- increases the available time to develop products within a technology generation by 50 per cent, from two years up to three." | 12:25 |
| kanzure | i'm not really sure what sort of planning usually goes into large chemistry projects | 12:26 |
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| fenn | this is not a "large chemistry project" | 12:30 |
| kanzure | go on? | 12:31 |
| fenn | it's only a few steps | 12:31 |
| kanzure | i wonder what the margin for error is on concentration at each step | 12:34 |
| fenn | i dont recally anyone linking to the actual POSAM paper so here it is http://www.ncbi.nlm.nih.gov/pmc/articles/PMC507883/ | 12:35 |
| fenn | .title | 12:35 |
| yoleaux | POSaM: a fast, flexible, open-source, inkjet oligonucleotide synthesizer and microarrayer | 12:35 |
| kanzure | "Reagents and waste are stored in glass bottles with GL-45 screw-top caps. Amber 500 ml bottles hold oxidizer and deprotection acid, while clear 2 l bottles hold acetonitrile and waste. Pressurizing nitrogen enters the reagent and solvent bottles through PTFE check valves and the waste bottle is under vacuum. Six PTFE solenoid valves (Model 190224S30, Angar, Florham Park, NJ) are used to control the flow of acetonitrile, oxidizer, acid, ... | 12:35 |
| kanzure | ... waste and other reagents." | 12:36 |
| CaptHindsight | fenn: just fyi the drop volume is now 1.5pL vs the 10pL of those earlier heads | 12:36 |
| CaptHindsight | so printed spot size will be smaller now as well | 12:37 |
| kanzure | "The inkjet oligonucleotide synthesis process uses standard phosphoramidite chemistry as described by [30], and modified for use in automated oligonucleotide synthesizers by [31]. Nucleoside phosphoramidites (bzdAMP, AcdCMP, ibudGMP, dTMP), 5-ethylthio-1H-tetrazole, and oxidizer (0.02 M iodine in pyridine/tetrahydrofuran/water) were purchased from Glen Research (Sterling, VA). Phosphoramidites and 5-ethylthio-1H-tetrazole were dissolved ... | 12:37 |
| kanzure | ... at 0.25 M in a mixture of 50% 3-methoxypropionitrile and 50% glutaronitrile (Sigma, St. Louis, MO). The solvents were dried for two days on molecular sieves before use. Synthesis-grade anhydrous acetonitrile and high-purity dichloromethane and dichloroacetic acid were purchased from Sigma. The detritylation solution used was 2.5% dichloroacetic acid in dichloromethane. Only 6 pl of phosphoramidite monomers and tetrazole were spotted ... | 12:37 |
| kanzure | ... on the slide using the inkjet print head; all other reagents were added 1 ml at a time using PTFE solenoid valves." | 12:37 |
| fenn | .wa diameter in microns of 1.5 picoliter sphere | 12:37 |
| yoleaux | sphere: volume 1.5 pL (picoliters): diameter in microns: 14.2 microns; Visual representation: http://is.gd/3hAnuA; Unit conversions: 0.56 mils; 5.6×10⁻⁴ inches; 14.2 µm (micrometers); 0.0142 mm (millimeters); 1.42×10⁻⁵ meters; Comparisons as length: ~0.5 × length of a skin cell (~30 µm); ~1.2 × oil filter mesh size (~450 microinches) | 12:38 |
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| fenn | .wa diameter in microns of 3 picoliter sphere | 12:39 |
| yoleaux | sphere: volume 3 pL (picoliters): diameter in microns: 10 3^(2/3) (2/pi)^(1/3) microns~~17.894 microns; Visual representation: http://is.gd/LXYfQX; Unit conversions: 0.7045 mils; 7.045×10⁻⁴ inches; 17.89 µm (micrometers); 0.01789 mm (millimeters); 1.789×10⁻⁵ meters; Comparisons as length: ~0.3 × length of a human sperm (~60 µm); ~0.6 × length of a skin cell (~30 µm); ~1.6 × oil filter mesh size (~450 microinches) | 12:39 |
| kanzure | they claim the chemistry is from: | 12:39 |
| kanzure | http://www.nature.com/nature/journal/v310/n5973/abs/310105a0.html | 12:39 |
| kanzure | and: | 12:39 |
| kanzure | http://www.ncbi.nlm.nih.gov/pubmed/3481013 | 12:39 |
| CaptHindsight | fenn: that is for a sphere, when the drop hist the tray the eventual spot size is based on its and the the trays surface tension | 12:40 |
| CaptHindsight | hist/hits | 12:40 |
| kanzure | https://www.google.com/patents/US5368823 | 12:40 |
| kanzure | https://www.google.com/patents/US5541314 | 12:40 |
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| fenn | i'm assuming it has a contact angle of 90 degrees so it would be a hemisphere, equivalent to 1/2 of a sphere with twice the volume | 12:40 |
| kanzure | oh look a cambrian genomics patent https://www.google.com/patents/WO2013126902A1 | 12:41 |
| kanzure | https://www.google.com/search?tbo=p&tbm=pts&hl=en&q=ininventor:%22Austen+HEINZ%22 | 12:41 |
| kanzure | hmm | 12:42 |
| fenn | did i mention i hate patents | 12:42 |
| kanzure | well i can't find the paper that they were using for their synthesis parameters | 12:42 |
| CaptHindsight | .wa diameter in microns of 20 picoliter sphere | 12:44 |
| yoleaux | sphere: volume 20 pL (picoliters): diameter in microns: 20 (15/pi)^(1/3) microns~~33.6778 microns; Visual representation: http://is.gd/hMkywP; Unit conversions: 1.326 mils; 0.001326 inches; 33.68 µm (micrometers); 0.03368 mm (millimeters); 3.368×10⁻⁵ meters; Comparisons as length: ~0.3 × length of a rod cell photoreceptor (~100 µm); ~0.6 × length of a human sperm (~60 µm); ~length of a skin cell (~30 µm) | 12:44 |
| CaptHindsight | but their 10pL drops of the actual fluids was ~150um | 12:46 |
| CaptHindsight | with a contact angle of around 45° | 12:46 |
| kanzure | we need one of the supplementary docs that runs through the exact reactions | 12:46 |
| delinquentme | Do we have anyone working with cell lines in wet labs? | 12:47 |
| CaptHindsight | " printing phosphoramidites before tetrazole, appears to help maintain virtual well integrity." | 12:47 |
| delinquentme | trying to get a borrowed copy of RPE-1 cells :D | 12:47 |
| fenn | whats the volume of a chord with 45 degree corners | 12:47 |
| CaptHindsight | "Changing the solvent ratio (3MP:MGN) from 1:1 to 1:3 also improved the integrity of the wells; however, the solutes are less soluble in the 1:3 3MP:MGN solvent and were more likely to clog inkjet nozzles." | 12:47 |
| delinquentme | something to do research / learning on prior to commercialization | 12:47 |
| CaptHindsight | Slides produced by the simplified surface-modification protocol described in Materials and methods improved our success rate to between 80-90%. This new modification process is reliable and trouble free, and is now the standard way we produce slides. | 12:48 |
| fenn | .c asin(3.14/4) | 12:48 |
| yoleaux | sin⁽⁻¹⁾(3.14/4) = 0.902696...; (result in radians) | 12:48 |
| fenn | .c sin(3.14/4) | 12:49 |
| yoleaux | sin(3.14/4) = 0.706825... | 12:49 |
| CaptHindsight | "In a separate project, we designed and constructed a temperature-ramping microarray scanner that, when used with POSaM produced slides, can monitor the dissociation kinetics of each reporter/target on the array, achieving single-base mismatch resolution. Besides the obvious use for SNP detection, the ability to synthesize any sequence of DNA and determine the apparent dissociation constant make it possible to test the algorithms | 12:50 |
| CaptHindsight | and heuristics used to select reporters, thereby increasing our understanding of how nucleic acids interact." | 12:50 |
| fenn | .wa diameter in microns of a 2*10/0.3 picoliter sphere | 12:50 |
| yoleaux | sphere: volume 2×10/0.3 pL (picoliters): diameter in microns: 50.31 microns; Visual representation: http://is.gd/pcTGUu; Unit conversions: 1.981 mils; 0.001981 inches; 50.31 µm (micrometers); 0.05031 mm (millimeters); 5.031×10⁻⁵ meters; Comparisons as length: ~0.4 × length of a dust mite (~125 µm); ~0.5 × length of a rod cell photoreceptor (~100 µm); ~0.84 × length of a human sperm (~60 µm) | 12:51 |
| fenn | well i dunno why it's 150 microns in diameter then | 12:51 |
| CaptHindsight | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC507883/figure/F5/ pics of the drops | 12:52 |
| fenn | anyway, is it possible to make smaller or larger drops by modifying the waveform of the printhead? | 12:53 |
| fenn | and will those drops end up in the same places as the other drops | 12:53 |
| CaptHindsight | and we don't really know if they used 10pL for certain, we don;t know if they actually measured the drop volume | 12:54 |
| kanzure | the posam operation manual says they buy "premixed" chemicals because it's sort of standardized already. hrm. | 12:54 |
| CaptHindsight | the native drop volume is ~1.5pL | 12:54 |
| CaptHindsight | the heads are capable of printing grey scale which means printing 1.5pL drops ina short time and having those drops combine into larger drops | 12:55 |
| fenn | since the head is moving i figured sequential drops would not overlap | 12:56 |
| CaptHindsight | it depends on how fast the head is moving | 12:57 |
| CaptHindsight | but thats how grey scale heads work | 12:57 |
| CaptHindsight | smaller drops combine in flight | 12:57 |
| fenn | how does that work | 12:57 |
| CaptHindsight | https://www.youtube.com/watch?v=SdkaE2rOt00 | 12:59 |
| kanzure | .title | 13:00 |
| yoleaux | JetXpert - Xaar 1001 Grayscale - YouTube | 13:00 |
| CaptHindsight | https://www.fujifilmusa.com/shared/bin/VersaDrop_TechReview.pdf an example | 13:01 |
| fenn | actually measuring the drops in the images with calipers they are more like 75-115 microns | 13:01 |
| fenn | in the posam paper | 13:02 |
| fenn | so for drop combining the later droplets are driven harder and thus go faster than the earlier droplets? | 13:03 |
| fenn | in that video the first droplet stayed separate but the 2nd 3rd and 4th droplets all combined | 13:04 |
| CaptHindsight | just an example | 13:05 |
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| CaptHindsight | to make things more complicated piezo heads have patents on the type of mode used for actuation | 13:05 |
| CaptHindsight | so the head makers all do it a bit differently | 13:06 |
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| CaptHindsight | my point was that you can make different drop sizes but how is dependent on the design of the head | 13:07 |
| CaptHindsight | I have to watch what I say since I'm under several NDA's with printhead makers | 13:08 |
| CaptHindsight | most piezo printheads have less than 1.5 degrees variance from nozzle to nozzle in drop accuracy | 13:10 |
| CaptHindsight | so drops will end up on top of each other but the surface tension of the drop and what it hits will determine the spot size | 13:11 |
| fenn | it's more important that they're repeatable enough to overlap every time | 13:12 |
| CaptHindsight | I have 10um for repeatability on the spec for the stage | 13:13 |
| fenn | if one drop misses then there are a whole bunch of oligos with the same deletion mutation, and that messes with the purification/error rejection | 13:13 |
| CaptHindsight | 282um is the native nozzle pitch | 13:14 |
| fenn | can spread out the risk by doing multiple copies of the same oligo though | 13:14 |
| CaptHindsight | if you want a different pitch is slows down the printing since you have to make more passes | 13:14 |
| CaptHindsight | 1/90th inch | 13:15 |
| fenn | so i guess 282um should be the horizontal pitch as well | 13:15 |
| CaptHindsight | doesn't have to be | 13:16 |
| CaptHindsight | could be 141um if the spots are under say 100um | 13:17 |
| fenn | you mean doing overlapping passes? or just having different horizontal/vertical pitches | 13:17 |
| CaptHindsight | then you make two passes to get drops spaced 141um | 13:18 |
| CaptHindsight | either | 13:18 |
| fenn | ok | 13:18 |
| fenn | i figure cross-contamination is more important to avoid than getting more spots on a plate | 13:18 |
| fenn | but that will have to be borne out by testing and measurement | 13:19 |
| CaptHindsight | they found a low cost silane to coat the slides | 13:19 |
| CaptHindsight | Rain-X | 13:19 |
| CaptHindsight | since we really can't modify the fluids much that are jetted | 13:20 |
| CaptHindsight | but we can play a bit with the surface tension of the slide coating | 13:20 |
| CaptHindsight | then again I'm not certain of the base fluids | 13:20 |
| CaptHindsight | maybe there are inert materials that we can add to modify surface tension | 13:21 |
| CaptHindsight | looks like POSaM just got lucky | 13:21 |
| fenn | so this versadrop thing just uses a longer pulse to get a longer jet and thus a larger drop | 13:23 |
| fenn | i'd rather do it that way to get a consistent drop velocity to ensure they all end up in the same location, regardless of size | 13:25 |
| CaptHindsight | Each cycle of synthesis includes (1) printing, (2) washing, (3) oxidation, (4) washing, (5) detritylation, and (6) washing. The first step of the reaction consists of phosphoramidite monomer printing followed by tetrazole printing, followed by a 2 min incubation. This first 3' base was reprinted to ensure a high density of oligonucleotide synthesis. Oxidization and detritylation steps were also carried out for 2 min each. | 13:25 |
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| fenn | CaptHindsight: is it possible to measure droplet velocity with the laser or is that just inferred from the location of the resulting spot? | 13:31 |
| CaptHindsight | fenn: they use the laser to detect diffraction of the light through the drops | 13:32 |
| fenn | but there is a delay between when you fire the print head and when you detect a droplet | 13:33 |
| CaptHindsight | we use a high speed camera system and strobe light to measure drop velocity | 13:33 |
| fenn | i guess what i want to know is what the signal on the droplet detector looks like... is it a sharp edged waveform or is it some noisy blob | 13:34 |
| CaptHindsight | Epson are slow 1-3m/sec | 13:34 |
| CaptHindsight | whats the beam diameter? | 13:35 |
| fenn | you tell me! | 13:35 |
| CaptHindsight | figure 2m/sec drop velocity | 13:35 |
| CaptHindsight | how long will the drops be in that beam? | 13:36 |
| fenn | the different nozzles have different chemicals and presumably different viscosities so i want to make sure that these all end up in the same place | 13:36 |
| kanzure | can you find me evidence that they are using evaporation on purpose | 13:37 |
| CaptHindsight | best way is to stop the head and fire | 13:37 |
| CaptHindsight | the same fluid should jet similarly each time fired | 13:38 |
| fenn | ok so if the beam diameter is 1mm and the droplet is 20 microns and going 2m/s it will be in the beam for (1mm+20micron)/(2m/s) = 510 microsec | 13:38 |
| CaptHindsight | we have to see how the different fluids behave in flight | 13:38 |
| CaptHindsight | there might be different top speeds for each fluid | 13:38 |
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| Infinityhf | Anyone here guide me? | 13:39 |
| kanzure | no | 13:39 |
| CaptHindsight | kanzure: a nice flow chart would have been handy in the POSaM docs | 13:39 |
| fenn | is stopping to fire a viable solution? there are supposedly 12inch/141um = 2162 stops per pass | 13:41 |
| fenn | that's a lot of wear and tear | 13:41 |
| Infinityhf | I cry | 13:42 |
| CaptHindsight | also have to look at the time between each print pass | 13:42 |
| CaptHindsight | after printing one fluid, how long is it until the next fluid is placed on top of it | 13:42 |
| fenn | its something like 2 minutes per reaction | 13:42 |
| CaptHindsight | wasn't clear to me | 13:43 |
| fenn | so 30 seconds until the next droplet is placed on top | 13:43 |
| kanzure | i think that if you time everything right then by the time you print the last drop for a layer, it's been 2 minutes for the first drop that was printed back at the start position | 13:43 |
| fenn | in one of the source files there's a "wait 30 seconds" at least | 13:43 |
| CaptHindsight | I didn't dig into making flow charts and timing estimates since I though that all this was already thought throug | 13:43 |
| CaptHindsight | *through | 13:44 |
| kanzure | sorry for misleading you | 13:44 |
| fenn | it's been thought through, just not by me :) | 13:44 |
| CaptHindsight | had a feeling but ........ | 13:44 |
| CaptHindsight | heh | 13:44 |
| CaptHindsight | I'll be back later | 13:44 |
| CaptHindsight | let me know what you decide to do | 13:45 |
| CaptHindsight | I can also look for linear servos and get the repeatability down to 1um and make provisions for much better printheads in the future | 13:46 |
| CaptHindsight | but this also makes the design more difficult for others to follow | 13:47 |
| CaptHindsight | at least at low cost | 13:47 |
| fenn | your ideas of "low cost" are already way beyond mine | 13:48 |
| fenn | i still dont get what's wrong with just hacking a printer | 13:48 |
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| fenn | "If nozzle failures are detected, the software reschedules the motion and firing of the printing head so that the most efficient printing path is taken." snazzy | 13:51 |
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| ParahSailin | problem? | 14:07 |
| nmz787_i | kanzure: oligos won't evaporate | 14:14 |
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| nmz787_i | generally the starting point is like some carboxyl on the bead, that gets primed chemicall to look like a nucleotide-end | 14:15 |
| nmz787_i | sometimes ppl don't use beads, and instead use other soluble molecules like PEG | 14:15 |
| kanzure | yes, we still need to actually use that | 14:15 |
| nmz787_i | i don't think there's any argon blow gun, unless it's near the parking area, to clean the nozzles | 14:16 |
| kanzure | so you never drain the wash cycles? | 14:16 |
| nmz787_i | an the solid-support (bead) is usually only used because they want to wash the unused nucleotide away... this isn't really a problem if you under-dose the reaction (under stoichiometric, and ensure all reagent is used) | 14:17 |
| fenn | there is a blow-dry step to remove the wash solvent | 14:19 |
| fenn | also argon is never mentioned anywhere, they use nitrogen for everything | 14:20 |
| fenn | nmz787_i: that is the worst advice ever | 14:21 |
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| fenn | you do realize we're going for >99.9% yield right | 14:22 |
| fenn | in each addition step, all oligos must be extended or else you get a deletion mutation in that strand | 14:24 |
| fenn | if you don't add enough reagent to ensure that every strand has a base added to it, you're just increasing the error rate unnecessarily | 14:25 |
| fenn | higher error rate makes it harder to purify and limits the length of oligo you can make | 14:26 |
| fenn | also it reduces the total quantity of DNA produced | 14:26 |
| fenn | you need an excess of reagent at every step. period. | 14:27 |
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| nmz787_i | I think with the microarrays they might just link to the surface of the ship | 14:29 |
| nmz787_i | chip* | 14:29 |
| nmz787_i | fenn: deletion shouldn't matter if you have some hybridization enrichment that relies on ligase | 14:38 |
| nmz787_i | but again, I was giving an example of liquid-phase techniques | 14:39 |
| nmz787_i | actually with the liquid phase stuff, they use a soluble support that they can crash/crystallize out, instead of a wash step | 14:39 |
| kanzure | fenn: it would be nice to have useful order-of-magnitude how much excess reagent is needed to be within "safety limits" for yield goals | 14:42 |
| kanzure | regarding argon and nitrogen, it's the abi 391 that uses argon everywhere and it's posam that uses nitrogen | 14:44 |
| nmz787_i | ah | 14:44 |
| nmz787_i | they're likely interchangable, as long as they're pure | 14:44 |
| nmz787_i | I wonder how different common contaminates, or for that matter the solvents/carriers are, solubilities differ for the two gasses | 14:45 |
| kanzure | so i thought it had to be an unreactive noble gas | 14:45 |
| nmz787_i | I think it just has to not interfere, not soak up contaminants too much (and maybe not be too attractive to absorb solvent) | 14:45 |
| kanzure | ok well maybe blowing anything at all is enough | 14:46 |
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| nmz787_i | except oxygen or steam | 14:47 |
| fenn | CO2 maybe changes the pH or whatever it's called when it's a non-water solvent | 14:48 |
| kanzure | .tw https://twitter.com/kaimtodner/status/621373610624741376 | 14:48 |
| yoleaux | Laurie love arrested on three usa extradition warrants. Granted bail this afternoon. (@kaimtodner) | 14:48 |
| fenn | doh | 14:48 |
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| gnusha | https://secure.diyhpl.us/cgit/diyhpluswiki/commit/?id=827eeccb Bryan Bishop: oligonucleotide synthesis notes >> http://diyhpl.us/diyhpluswiki/dna/synthesis/notes/ | 15:31 |
| kanzure | http://diyhpl.us/wiki/dna/synthesis/notes/ | 15:32 |
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| CaptHindsight | fenn: hacking a printer will not get you close to the repeatability require for multilayers of drops in the same spots | 15:54 |
| CaptHindsight | fenn: the t-shirt printers hack the Epsons but that is about all that they are good for, making something that looks like an image or a graphic | 15:55 |
| CaptHindsight | nmz787_i: Polyethylene glycol = (PEG)? | 15:58 |
| fenn | well at minimum it can deposit spots that are large enough to be visible | 15:58 |
| CaptHindsight | the dimatix system not even this size was well over $50K | 16:00 |
| fenn | could i interest you in a gold plated turd | 16:01 |
| CaptHindsight | fenn: http://www.fujifilmusa.com/products/industrial_inkjet_printheads/deposition-products/dmp-2800/ ~$30k | 16:03 |
| fenn | it's an inkjet printer with 2 cameras and a vacuum chuck | 16:04 |
| CaptHindsight | they just use 2 linear servos | 16:04 |
| CaptHindsight | https://www.epson.com/cgi-bin/Store/jsp/Pro/SeriesSureColorF2000/Overview.do?UseCookie=yes $20K | 16:08 |
| nmz787_i | CaptHindsight: PEG == polyethylene glycol | 16:08 |
| fenn | someone ported falstad's circuit simulator to javascript http://lushprojects.com/circuitjs/circuitjs.html | 16:08 |
| kanzure | "Because coupling is not always quantitative, a small percentage (up to 2%) of support-bound nucleotides can fail to undergo addition." | 16:09 |
| kanzure | ..... 2% | 16:09 |
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| kanzure | just typed this | 16:19 |
| kanzure | http://diyhpl.us/~bryan/papers2/DNA/abi391/chemistry.txt | 16:19 |
| CaptHindsight | so do we need to couple the first base to the sample tray/slide? | 16:31 |
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| CaptHindsight | couple/bond/adhere/make sticky etc | 16:34 |
| fenn | you just typed that eh | 16:35 |
| CaptHindsight | The Model 391 DNA Synthesizer uses a solid phase synthesis chemistry in which the growing DNA chain remains covalently attached to an insoluble support. All reagents and solvents flow through the support which is contained within a synthesis column. The support used for DNA synthesis is Controlled-Pore-Glass (CPG) | 16:48 |
| CaptHindsight | http://imagebin.ca/v/28slSKg3GYo0 391 block diagram | 16:53 |
| CaptHindsight | the support column contains the Controlled Pore Glass (CPG) | 16:54 |
| CaptHindsight | they pump the different bases and reagents through the porous glass layer by layer | 16:55 |
| CaptHindsight | CPG has a linker attached to its surface via a siloxane bond | 16:56 |
| CaptHindsight | http://diyhpl.us/~bryan/papers2/DNA/abi391/ABI%20391-manual.pdf Chapter 6 has the chemistry | 16:57 |
| CaptHindsight | Chapter 5 shows how it physically works from a hydraulics point of view | 16:58 |
| kanzure | yes, the oligo needs to be attached to the surface or attached to a bead which doesn't move (without the inkjet knowing where to print) | 17:12 |
| kanzure | .wik 5-HTTLPR | 17:13 |
| yoleaux | "5-HTTLPR (serotonin-transporter-linked polymorphic region) is a degenerate repeat polymorphic region in SLC6A4, the gene that codes for the serotonin transporter. Since the polymorphism was identified in the middle of the 1990s, it has been extensively investigated, e.g., in connection with neuropsychiatric disorders." — https://en.wikipedia.org/wiki/5-HTTLPR | 17:13 |
| CaptHindsight | kanzure: ok, so now the oligo doesn't get washed, rinsed. blown, drawn, shaken away since it is anchored by the first base | 17:16 |
| kanzure | i am attempting to fully emulate the reaction in my head, but still assembling details of why this is expected to work, etc. and also making sure that all of the reagents have been specified. | 17:18 |
| kanzure | also i think this might mean that we may want the printhead to deposit the initial linkers....? | 17:19 |
| kanzure | there was a long "slide prep" section in the posam docs... not all of those slide prep steps can be done on the printhead :-(. | 17:19 |
| kanzure | (it wasn't just "apply rainx") | 17:19 |
| drethelin | hmm | 17:27 |
| drethelin | what's the best probiotic | 17:27 |
| drethelin | like if I assume my gut flora is probably somewhat fucked up | 17:28 |
| drethelin | and I want to replace it with something less shitty | 17:28 |
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| rigel | ill sell you my poop. you can transplant it on your own. | 17:36 |
| CaptHindsight | how do we know that this is good quality poop? | 17:37 |
| rigel | trust me, it's straight from the tap | 17:38 |
| CaptHindsight | not just crap that you can find anywhere? | 17:38 |
| CaptHindsight | so you're full of crap and willing to sell | 17:39 |
| rigel | franchises available | 17:40 |
| CaptHindsight | picturing the possible logos | 17:41 |
| fenn | the printhead should deposit the initial linkers and the spots should be smaller than all of the other spots | 17:41 |
| CaptHindsight | kanzure: the slides go through several cleaning and drying steps | 17:42 |
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| kanzure | fenn: ah, so then we need to graduate to two printheads for that | 18:08 |
| kanzure | because we used up the other channels already | 18:08 |
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| nmz787_i | CaptHindsight: the initial slide 'functionalization' steps are laid out in the section "In-House Epoxysilane Slide Preparation" | 18:30 |
| nmz787_i | I found it here http://www.genomebiology.com/content/supplementary/gb-2004-5-8-r58-s2.pdf | 18:31 |
| nmz787_i | but I think it's also on the posam site | 18:31 |
| nmz787_i | "That said, we have had partial success using epoxysilane slides supplied by Bioslide (Walnut, CA) and Xenopore (Hawethorne, NJ). For users wishing to experiment with these or other commercial slide types, we recommend storage in sealed vapor-barrier bags (the anti-static type used to ship electronics components work very well) containing desiccant packs with indicator. Slides should be stored in a cool, dry location. Since epoxy is reactive to | 18:32 |
| nmz787_i | primary amines, epoxysilane slides should not be stored in the same Desiccator as amino-modified slides. Generally, commercial slides should be pre-treated and handled according to the guidelines used for the in-house slides." | 18:32 |
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| CaptHindsight | 6 channel Epson heads are <$200 ea, 8 channel are ~$450 | 18:53 |
| CaptHindsight | Epson recently started adding a small micro into some of the heads for DRM | 18:55 |
| CaptHindsight | they don't want the lower cost heads being installed in place of the more expensive heads in their printers | 18:55 |
| CaptHindsight | I think the 8 channel unlocked heads are ~$600ea | 18:57 |
| CaptHindsight | synthesis is achieved by using a reactive epoxysilane capable of covalent attachment to the | 18:58 |
| CaptHindsight | silanol groups on the glass surface in a background of longer-chain silanes which also attach to | 18:58 |
| CaptHindsight | the glass but provide the hydrophobic properties and do not participate in base attachment | 18:58 |
| CaptHindsight | during synthesis. These hydrophobic silanes are obtained by using RainX. Yes, RainX | 18:58 |
| CaptHindsight | so the silane covalent bonds to the glass slide | 19:00 |
| CaptHindsight | the silane is also hydrophobic | 19:00 |
| CaptHindsight | and does not participate in the base attachment during synthesis, so what does this mean? | 19:01 |
| CaptHindsight | base attachment to the slide or the bases attaching to each other? | 19:02 |
| CaptHindsight | I don't see where the first base is bonded to anything | 19:07 |
| CaptHindsight | but a photocleavable linker could be added if we want that | 19:11 |
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| CaptHindsight | http://www.pastebin.ca/3063720 The following playbook file describes the standard synthesis procedure. | 19:14 |
| CaptHindsight | Arrayer File Formats http://www.pastebin.ca/3063723 | 19:17 |
| CaptHindsight | http://imagebin.ca/v/28tTbEgeiPCD maybe we can reach a consensus on deciphering the steps | 19:19 |
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| kanzure | their playbook file is useless | 19:53 |
| CaptHindsight | does it make any sense to you? | 19:53 |
| kanzure | yes, but it's also useless.... | 19:53 |
| CaptHindsight | is it just garbage | 19:53 |
| CaptHindsight | the epoxy on the slides is reactive to amines | 19:54 |
| kanzure | it's mostly garbage. the actual amount of time to wait is given in some of the other documents, etc. the reaction steps are also given in the other documents. | 19:55 |
| CaptHindsight | so maybe the first base does bond to the epoxy | 19:56 |
| CaptHindsight | and the silane adds the hydrophobic surface tension modifier bit | 19:58 |
| CaptHindsight | I've been wanting to explore epoxy silanes and urethane silanes for radcure | 19:59 |
| CaptHindsight | or hybrid radcure | 20:00 |
| CaptHindsight | The cost for the reagents is low (less than US$50 per slide, see Table Table1)1) as such tiny amounts of phosphoramidite and tetrazole are required. | 20:08 |
| CaptHindsight | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC507883/table/T1/ | 20:09 |
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| CaptHindsight | zzzzzzz | 20:20 |
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| kanzure | that is a good table | 21:10 |
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| kanzure | .title https://www.youtube.com/watch?v=fFueP7rDnDw | 21:40 |
| yoleaux | Phosphodiesters: Phosphorus in Nucleic Acids - YouTube | 21:40 |
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| --- Log closed Fri Jul 17 00:00:13 2015 | ||
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