--- Log opened Thu Jul 16 00:00:12 2015
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03:05 < archels_> kanzure: G-protein coupled optogenetics sounds relatively slow. Did they combine it with the usual opsins or as a replacement thereof?
03:06 < archels_> woah, even slower that I thought. Timescale of seconds.
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07:16 < kanzure> .title http://www.c2o.pro.br/en/automation/x73.html
07:16 < yoleaux> Building a Respirometer using FSM, Tcl and Arduino
07:17 < kanzure> "lab devices with rs232 interfaces" http://www.c2o.pro.br/en/automation/x45.html
07:17 < kanzure> indiebio: hi
07:22 < kanzure> .title https://news.ycombinator.com/item?id=9894570
07:22 < yoleaux> 1.5 TB of Dark Net Market scrapes | Hacker News
07:39 < indiebio> hi kanzure
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07:52 < kanzure> stopping a comet http://web.archive.org/web/20140415063820/http://szabo.best.vwh.net/comet.thread.html
07:53 < kanzure> self-replication stuff http://szabo.best.vwh.net/nano.musings.html
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09:12 < kanzure> curious approach to keeping track of stack depth for context switching https://news.ycombinator.com/item?id=9896462
09:13 < kanzure> http://faq.sealedabstract.com/uninterruptible_programming_supply/
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09:18 < kanzure> long conference talk on adhd https://www.youtube.com/watch?v=SCAGc-rkIfo
09:22 < 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
09:28 < kanzure> working memory defecits https://www.youtube.com/watch?v=SCAGc-rkIfo&t=19m40s
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09:42 < nmz787_i> kanzure: any ideas for bonding a top-plate to a nano-channel between 2 microchannels?
09:43 < kanzure> does the bonding problem change because of the channels...?
09:47 < 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
09:48 < 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
09:49 < nmz787_i> I guess I could get some cover slip glass characterized with an interferometer
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09:50 < nmz787_i> or maybe at that scale the FIB/SEM would be fine
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09:56 < 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
09:57 < nmz787_i> maybe 40nm?
09:57 < nmz787_i> https://hal.archives-ouvertes.fr/jpa-00229994/document
09:58 < 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
09:59 < 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."
10:01 < kanzure> adhd is time travel https://www.youtube.com/watch?v=SCAGc-rkIfo&t=1h12m50s
10:07 < CaptHindsight> unless the chamber is beyond repair that ion beam is a good deal
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10:22 < kanzure> i think they hired richard dreyfuss for this lecture
10:25 < CaptHindsight> was a home found for the inkjet?
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10:28 < kanzure> CaptHindsight: yep, los angeles biohackers
10:28 < kanzure> or nmz787
10:29 < CaptHindsight> nmz787: are you in the LA area?
10:30 < CaptHindsight> http://www.biohackers.la/  this group?
10:39 < kanzure> yes that group
10:39 < kanzure> nmz787 is in portland, oregon
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10:42 < fenn> wiki.biohackers.la is down so it's hard to judge how active the group is
10:43 < fenn> "Ignore the smell - it's not us."
10:55 < CaptHindsight> http://www.meetup.com/LAbiohackers/
10:56 < kanzure> didn't we backup their wiki at some point
10:56 < kanzure> was that a thing we did?
11:00 < CaptHindsight> reading through the member list they have some actual biologists
11:01 < 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
11:02 < 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^
11:03 < heath> just thought i'd toss that in the mix
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11:06 < fenn> ok so it looks like la biohackers actually have events and people attend those events
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11:08 < 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
11:16 < juri_> that's a problem at BUGS, too.
11:18 < 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
11:19 < 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
11:20 < 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
11:21 < juri_> fenn: good point. thanks. ;)
11:22 < fenn> kanzure: what happened with the lab in carlsbad?
11:23 < 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
11:24 < kanzure> i believe he is just renting out benches
11:24 < fenn> benches do not a lab make
11:25 < kanzure> well, there's equipment of course
11:31 < 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
11:34 < 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)
11:36 < kanzure> 50 micron diameter, not bad
11:37 < kanzure> maybe we should put it in a trailer and just buy a truck
11:37 < fenn> you're missing the point
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11:38 < 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
11:39 < kanzure> machine design services
11:39 < CaptHindsight> what would they tweak?
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11:39 < CaptHindsight> what in the machine design needs tweaking?
11:40 < 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
11:41 < 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
11:42 < 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, ..
11:43 < kanzure> mechanics are easy
11:43 < kanzure> enclosure leaks...
11:43 < kanzure> finding solvents compatible with the mechanical components
11:44 < 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"
11:45 < CaptHindsight> run cleaning cycles
11:45 < kanzure> hah maybe we should just email them, sales@azcobiotech.com
11:46 < 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
11:47 < CaptHindsight> yeah, we've been around this circle before
11:48 < kanzure> yield/error rate is different from "oshit i don't have any oligos whatsoever"
11:49 < CaptHindsight> you have to be able to print the bases and the chems to links them, that is the first step
11:50 < fenn> (btw the htsresources link was related somehow to biotechnbeyond)
11:50 < CaptHindsight> then you learn how to link them into longer sections
11:51 < 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
11:52 < 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
11:53 < CaptHindsight> argon blaster vs argon positive pressure
11:54 < 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
11:55 < kanzure> don't worry about the software
11:55 < fenn> so, what's a typical linker for starting an oligo?
11:56 < 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
11:57 < 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.
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11:58 < 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
11:59 < 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
12:00 < 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
12:01 < 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
12:02 < 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
12:03 < 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?
12:04 < 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
12:05 < 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
12:06 < 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?
12:07 < 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
12:10 < kanzure> we need a chemist
12:11 < CaptHindsight> need a linker?
12:12 < 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
12:14 < 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
12:15 < 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
12:17 < 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)
12:19 < 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
12:20 < 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
12:23 < kanzure> i mean electron availability
12:25 < 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."
12:26 < kanzure> i'm not really sure what sort of planning usually goes into large chemistry projects
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12:30 < fenn> this is not a "large chemistry project"
12:31 < kanzure> go on?
12:31 < fenn> it's only a few steps
12:34 < kanzure> i wonder what the margin for error is on concentration at each step
12:35 < 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, ...
12:36 < 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
12:37 < 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
12:38 < 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)
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12:39 < 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
12:40 < 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
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12:40 < 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
12:41 < 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
12:42 < 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
12:44 < 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)
12:46 < 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
12:47 < 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
12:48 < 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)
12:49 < fenn> .c sin(3.14/4)
12:49 < yoleaux> sin(3.14/4) = 0.706825...
12:50 < 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
12:51 < 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
12:52 < CaptHindsight> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC507883/figure/F5/  pics of the drops
12:53 < 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
12:54 < 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
12:55 < 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
12:56 < fenn> since the head is moving i figured sequential drops would not overlap
12:57 < 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
12:59 < CaptHindsight> https://www.youtube.com/watch?v=SdkaE2rOt00
13:00 < kanzure> .title
13:00 < yoleaux> JetXpert - Xaar 1001 Grayscale - YouTube
13:01 < 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
13:02 < fenn> in the posam paper
13:03 < fenn> so for drop combining the later droplets are driven harder and thus go faster than the earlier droplets?
13:04 < fenn> in that video the first droplet stayed separate but the 2nd 3rd and 4th droplets all combined
13:05 < CaptHindsight> just an example
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13:05 < CaptHindsight> to make things more complicated piezo heads have patents on the type of mode used for actuation
13:06 < CaptHindsight> so the head makers all do it a bit differently
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13:07 < CaptHindsight> my point was that you can make different drop sizes but how is dependent on the design of the head
13:08 < CaptHindsight> I have to watch what I say since I'm under several NDA's with printhead makers
13:10 < CaptHindsight> most piezo printheads have less than 1.5 degrees variance from nozzle to nozzle in drop accuracy
13:11 < 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
13:12 < fenn> it's more important that they're repeatable enough to overlap every time
13:13 < 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
13:14 < 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
13:15 < CaptHindsight> 1/90th inch
13:15 < fenn> so i guess 282um should be the horizontal pitch as well
13:16 < CaptHindsight> doesn't have to be
13:17 < 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
13:18 < 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
13:19 < 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
13:20 < 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
13:21 < CaptHindsight> maybe there are inert materials that we can add to modify surface tension
13:21 < CaptHindsight> looks like POSaM just got lucky
13:23 < fenn> so this versadrop thing just uses a longer pulse to get a longer jet and thus a larger drop
13:25 < 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.
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13:31 < fenn> CaptHindsight: is it possible to measure droplet velocity with the laser or is that just inferred from the location of the resulting spot?
13:32 < CaptHindsight> fenn: they use the laser to detect diffraction of the light through the drops
13:33 < 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
13:34 < 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
13:35 < CaptHindsight> whats the beam diameter?
13:35 < fenn> you tell me!
13:35 < CaptHindsight> figure 2m/sec drop velocity
13:36 < 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
13:37 < 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
13:38 < 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
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13:39 < 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
13:41 < 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
13:42 < 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
13:43 < 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
13:44 < 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
13:45 < CaptHindsight> let me know what you decide to do
13:46 < 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
13:47 < CaptHindsight> but this also makes the design more difficult for others to follow
13:47 < CaptHindsight> at least at low cost
13:48 < 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
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13:51 < 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
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14:07 < ParahSailin> problem?
14:14 < nmz787_i> kanzure: oligos won't evaporate
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14:15 < 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
14:16 < 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?
14:17 < 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)
14:19 < fenn> there is a blow-dry step to remove the wash solvent
14:20 < fenn> also argon is never mentioned anywhere, they use nitrogen for everything
14:21 < fenn> nmz787_i: that is the worst advice ever
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14:22 < fenn> you do realize we're going for >99.9% yield right
14:24 < fenn> in each addition step, all oligos must be extended or else you get a deletion mutation in that strand
14:25 < 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
14:26 < 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
14:27 < fenn> you need an excess of reagent at every step. period.
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14:29 < nmz787_i> I think with the microarrays they might just link to the surface of the ship
14:29 < nmz787_i> chip*
14:38 < nmz787_i> fenn: deletion shouldn't matter if you have some hybridization enrichment that relies on ligase
14:39 < 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
14:42 < 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
14:44 < 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
14:45 < 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)
14:46 < kanzure> ok well maybe blowing anything at all is enough
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14:47 < nmz787_i> except oxygen or steam
14:48 < 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
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15:31 < gnusha> https://secure.diyhpl.us/cgit/diyhpluswiki/commit/?id=827eeccb Bryan Bishop: oligonucleotide synthesis notes >> http://diyhpl.us/diyhpluswiki/dna/synthesis/notes/
15:32 < kanzure> http://diyhpl.us/wiki/dna/synthesis/notes/
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15:54 < CaptHindsight> fenn: hacking a printer will not get you close to the repeatability require for multilayers of drops in the same spots
15:55 < 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
15:58 < CaptHindsight> nmz787_i: Polyethylene glycol = (PEG)?
15:58 < fenn> well at minimum it can deposit spots that are large enough to be visible
16:00 < CaptHindsight> the dimatix system not even this size was well over $50K
16:01 < fenn> could i interest you in a gold plated turd
16:03 < CaptHindsight> fenn: http://www.fujifilmusa.com/products/industrial_inkjet_printheads/deposition-products/dmp-2800/  ~$30k
16:04 < fenn> it's an inkjet printer with 2 cameras and a vacuum chuck
16:04 < CaptHindsight> they just use 2 linear servos
16:08 < 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
16:09 < 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%
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16:19 < kanzure> just typed this
16:19 < kanzure> http://diyhpl.us/~bryan/papers2/DNA/abi391/chemistry.txt
16:31 < CaptHindsight> so do we need to couple the first base to the sample tray/slide?
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16:34 < CaptHindsight> couple/bond/adhere/make sticky etc
16:35 < fenn> you just typed that eh
16:48 < 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)
16:53 < CaptHindsight> http://imagebin.ca/v/28slSKg3GYo0  391 block diagram
16:54 < CaptHindsight> the support column contains the Controlled Pore Glass (CPG)
16:55 < CaptHindsight> they pump the different bases and reagents through the porous glass layer by layer
16:56 < CaptHindsight> CPG has a linker attached to its surface via a siloxane bond
16:57 < CaptHindsight> http://diyhpl.us/~bryan/papers2/DNA/abi391/ABI%20391-manual.pdf  Chapter 6 has the chemistry
16:58 < CaptHindsight> Chapter 5 shows how it physically works from a hydraulics point of view
17:12 < 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)
17:13 < 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
17:16 < CaptHindsight> kanzure: ok, so now the oligo doesn't get washed, rinsed. blown, drawn, shaken away since it is anchored by the first base
17:18 < 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.
17:19 < 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")
17:27 < drethelin> hmm
17:27 < drethelin> what's the best probiotic
17:28 < 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
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17:36 < rigel> ill sell you my poop. you can transplant it on your own.
17:37 < CaptHindsight> how do we know that this is good quality poop?
17:38 < rigel> trust me, it's straight from the tap
17:38 < CaptHindsight> not just crap that you can find anywhere?
17:39 < CaptHindsight> so you're full of crap and willing to sell
17:40 < rigel> franchises available
17:41 < 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
17:42 < CaptHindsight> kanzure: the slides go through several cleaning and drying steps
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18:08 < 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
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18:30 < nmz787_i> CaptHindsight: the initial slide 'functionalization' steps are laid out in the section "In-House Epoxysilane Slide Preparation"
18:31 < 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
18:32 < 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."
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18:53 < CaptHindsight> 6 channel Epson heads are <$200 ea, 8 channel are ~$450
18:55 < 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
18:57 < CaptHindsight> I think the 8 channel unlocked heads are ~$600ea
18:58 < 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
19:00 < CaptHindsight> so the silane covalent bonds to the glass slide
19:00 < CaptHindsight> the silane is also hydrophobic
19:01 < CaptHindsight> and does not participate in the base attachment during synthesis, so what does this mean?
19:02 < CaptHindsight> base attachment to the slide or the bases attaching to each other?
19:07 < CaptHindsight> I don't see where the first base is bonded to anything
19:11 < CaptHindsight> but a photocleavable linker could be added if we want that
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19:14 < CaptHindsight> http://www.pastebin.ca/3063720  The following playbook file describes the standard synthesis procedure.
19:17 < CaptHindsight> Arrayer File Formats http://www.pastebin.ca/3063723
19:19 < CaptHindsight> http://imagebin.ca/v/28tTbEgeiPCD  maybe we can reach a consensus on deciphering the steps
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19:53 < 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
19:54 < CaptHindsight> the epoxy on the slides is reactive to amines
19:55 < 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.
19:56 < CaptHindsight> so maybe the first base does bond to the epoxy
19:58 < CaptHindsight> and the silane adds the hydrophobic surface tension modifier bit
19:59 < CaptHindsight> I've been wanting to explore epoxy silanes and urethane silanes for radcure
20:00 < CaptHindsight> or hybrid radcure
20:08 < 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.
20:09 < CaptHindsight> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC507883/table/T1/
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20:20 < CaptHindsight> zzzzzzz
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21:10 < kanzure> that is a good table
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21:40 < kanzure> .title https://www.youtube.com/watch?v=fFueP7rDnDw
21:40 < yoleaux> Phosphodiesters: Phosphorus in Nucleic Acids - YouTube
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--- Log closed Fri Jul 17 00:00:13 2015