Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot
Dear Joe -- Does anyone have information about how long it takes to set up a 96- well tray for the crystallization robots available? Besides cost per tray and maintenance cost, another important feature we consider is the time for setting up a 96-well tray. It is an important factor since we are talking about sub-microliter drops. The japanese protein-crystallization system (PXS) can handle 7680 drops / hour, from sample and solution disposal to sealing and incubator storage. You can have a look at the movie: http://pfweis.kek.jp/system/PXS.html or at the paper (PubMed 16929107). HTH. Kind regards. Leo Chavas Leonard, Ph.D. Research Associate Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: [EMAIL PROTECTED]
Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot
Actually, 3.5mins plate-to-plate for our 3-drop protocols (50:100, 75:75, 100:50ul) on our mosquito. (Not sure what protocol Artem is using, must have lots of tip changes.) Janet's reply reads slightly misleadingly: if you're concerned with plates/hour (which we are, since we have users show up with piles of 20-40 trays on Friday evenings), then mosquito seems(*) to be quickest by quite a bit, as it does not have a wash cycle: as soon as one plate is done, you wang on the next one. If you only do a plate at a time(**), then the wash cycle won't bother you, and phoenix is indeed as quick as mosquito. phx. (*) I must confess, though, I've not done extensive research recently. When I did poke around a year ago, the plate-to-plate timing was exceedingly hard to get out of sales-persons -- frustratingly. (**) Mind you, I'm not sure why you'd only do a plate at a time: protein variation is far more useful to explore than chemical variation. Artem Evdokimov wrote: About 2 – 2.5 minutes on Mosquito for single-drop protocol, scaled according to the number of drops per well (7 minutes for 3-drop trays). About 25 minutes on a Tecan-derived platform (numbers vary greatly depending on the particular configuration). Artem *From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf Of *JOE CRYSTAL *Sent:* Monday, April 14, 2008 5:10 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot Hi, Does anyone have information about how long it takes to set up a 96-well tray for the crystallization robots available? Besides cost per tray and maintenance cost, another important feature we consider is the time for setting up a 96-well tray. It is an important factor since we are talking about sub-microliter drops. Best, Joe On Fri, Jan 18, 2008 at 12:28 PM, Lisa A Nagy [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] wrote: Al's Oil on the plates: What a nightmare!!! The oil creeps up the plate and over the sides. It dissolves adhesives. It makes me say bad words in multiple languages. Bigger drops + no oil = fewer bad words. Lisa -- Lisa A. Nagy, Ph.D. University of Alabama-Birmingham [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Patrick Shaw Stewart Sent: Friday, January 18, 2008 2:20 AM To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fwd: [ccp4bb] crystallisation robot One thing that people often overlook is that quite a lot of protein can be lost by denaturation on the surface of the drop. This is more significant for smaller drops. Two suggestions: (1) increase the proportion of protein in the - technical term - teeny drop to say two thirds and (2) cover the drops with oil eg Al's oils (silicone/paraffin). You still get vapor diffusion though the oil , and you'd like to slow up equilibration. of course (2) slows up the robotics a little, but both should be trivial to set up..
Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot
Thanks Leo for your introduction. However, the link is broken. Please look at the following page. http://pfweis.kek.jp/protein/Robot/PXS/index-e.html Best regards, Yusuke --- Yusuke Yamada, Ph.D. Photon Factory High Energy Accelerator Research Organization [EMAIL PROTECTED] --- Chavas Leo wrote: Dear Joe -- Does anyone have information about how long it takes to set up a 96-well tray for the crystallization robots available? Besides cost per tray and maintenance cost, another important feature we consider is the time for setting up a 96-well tray. It is an important factor since we are talking about sub-microliter drops. The japanese protein-crystallization system (PXS) can handle 7680 drops / hour, from sample and solution disposal to sealing and incubator storage. You can have a look at the movie: http://pfweis.kek.jp/system/PXS.html or at the paper (PubMed 16929107). HTH. Kind regards. Leo Chavas Leonard, Ph.D. Research Associate Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: [EMAIL PROTECTED]
Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot
We use Cartesian Honeybee X8 machines (8 tips). They take about 10 minutes to set a 96-drop plate including the washes of the tips. 3 or 4 drops per condition wouldnt take much longer. Optimisation and additive/detergent screens take a little less time. The plates are pipetted under a close-fitting cover to (virtually) eliminate evaporation, which IMO is better than a humidity chamber. Consumable costs extend to isopropanol and water, with the occasional replacement valve or tip. Since people here also tend to turn up at beer o'clock on a Friday evening (must be an Oxford thing...) we have two machines (and another one imminent) to increase throughput. HTH Tom ** Tom Walter B.Sc. M.Res. ** ** Oxford Protein Production FacilityTel: +44 (0)1865 287747 ** ** Wellcome Trust Centre for Human Genetics Fax: +44 (0)1865 287547 ** ** Roosevelt Drive [EMAIL PROTECTED] ** ** Headington, Oxford OX3 7BNhttp://www.oppf.ox.ac.uk ** Original message Date: Mon, 14 Apr 2008 17:10:26 -0400 From: JOE CRYSTAL [EMAIL PROTECTED] Subject: Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot To: CCP4BB@JISCMAIL.AC.UK Hi, Does anyone have information about how long it takes to set up a 96-well tray for the crystallization robots available? Besides cost per tray and maintenance cost, another important feature we consider is the time for setting up a 96-well tray. It is an important factor since we are talking about sub-microliter drops. Best, Joe On Fri, Jan 18, 2008 at 12:28 PM, Lisa A Nagy [EMAIL PROTECTED] wrote: Al's Oil on the plates: What a nightmare!!! The oil creeps up the plate and over the sides. It dissolves adhesives. It makes me say bad words in multiple languages. Bigger drops + no oil = fewer bad words. Lisa -- Lisa A. Nagy, Ph.D. University of Alabama-Birmingham [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Patrick Shaw Stewart Sent: Friday, January 18, 2008 2:20 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fwd: [ccp4bb] crystallisation robot One thing that people often overlook is that quite a lot of protein can be lost by denaturation on the surface of the drop. This is more significant for smaller drops. Two suggestions: (1) increase the proportion of protein in the - technical term - teeny drop to say two thirds and (2) cover the drops with oil eg Al's oils (silicone/paraffin). You still get vapor diffusion though the oil , and you'd like to slow up equilibration. of course (2) slows up the robotics a little, but both should be trivial to set up..
Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot
Off the top of my head: Phoenix Robot: ~2min (incl. reformatting) Hydra2plusOne: ~5min (incl. reformatting and washing of needles) Mosquito: ~2min (no reformatting, needles are disposable) Cartesian: ~20min (incl. washing) At 11:10 PM 4/14/2008, JOE CRYSTAL wrote: Hi, Does anyone have information about how long it takes to set up a 96-well tray for the crystallization robots available? Besides cost per tray and maintenance cost, another important feature we consider is the time for setting up a 96-well tray. It is an important factor since we are talking about sub-microliter drops. Best, Joe On Fri, Jan 18, 2008 at 12:28 PM, Lisa A Nagy mailto:[EMAIL PROTECTED][EMAIL PROTECTED] wrote: Al's Oil on the plates: What a nightmare!!! The oil creeps up the plate and over the sides. It dissolves adhesives. It makes me say bad words in multiple languages. Bigger drops + no oil = fewer bad words. Lisa -- Lisa A. Nagy, Ph.D. University of Alabama-Birmingham mailto:[EMAIL PROTECTED][EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Patrick Shaw Stewart Sent: Friday, January 18, 2008 2:20 AM To: mailto:CCP4BB@JISCMAIL.AC.UKCCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fwd: [ccp4bb] crystallisation robot One thing that people often overlook is that quite a lot of protein can be lost by denaturation on the surface of the drop. This is more significant for smaller drops. Two suggestions: (1) increase the proportion of protein in the - technical term - teeny drop to say two thirds and (2) cover the drops with oil eg Al's oils (silicone/paraffin). You still get vapor diffusion though the oil , and you'd like to slow up equilibration. of course (2) slows up the robotics a little, but both should be trivial to set up.. ___ Jochen Muller-Dieckmann, Ph.D. Team Leader EMBL Hamburg Outstation c/o DESY, Notkestr. 85 D-22603 Hamburg Germany Ph: +49-40-89902-201FAX: +49-40-89902-215 ___ http://www.embl-hamburg.de/services/crystallisation
Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot
Hi, Does anyone have information about how long it takes to set up a 96-well tray for the crystallization robots available? Besides cost per tray and maintenance cost, another important feature we consider is the time for setting up a 96-well tray. It is an important factor since we are talking about sub-microliter drops. Best, Joe On Fri, Jan 18, 2008 at 12:28 PM, Lisa A Nagy [EMAIL PROTECTED] wrote: Al's Oil on the plates: What a nightmare!!! The oil creeps up the plate and over the sides. It dissolves adhesives. It makes me say bad words in multiple languages. Bigger drops + no oil = fewer bad words. Lisa -- Lisa A. Nagy, Ph.D. University of Alabama-Birmingham [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Patrick Shaw Stewart Sent: Friday, January 18, 2008 2:20 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fwd: [ccp4bb] crystallisation robot One thing that people often overlook is that quite a lot of protein can be lost by denaturation on the surface of the drop. This is more significant for smaller drops. Two suggestions: (1) increase the proportion of protein in the - technical term - teeny drop to say two thirds and (2) cover the drops with oil eg Al's oils (silicone/paraffin). You still get vapor diffusion though the oil , and you'd like to slow up equilibration. of course (2) slows up the robotics a little, but both should be trivial to set up..
Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot
Does this include the customary grieving period? On Apr 14, 2008, at 2:10 PM, JOE CRYSTAL wrote: Hi, Does anyone have information about how long it takes to set up a 96- well tray for the crystallization robots available? Besides cost per tray and maintenance cost, another important feature we consider is the time for setting up a 96-well tray. It is an important factor since we are talking about sub-microliter drops. Best, Joe On Fri, Jan 18, 2008 at 12:28 PM, Lisa A Nagy [EMAIL PROTECTED] wrote: Al's Oil on the plates: What a nightmare!!! The oil creeps up the plate and over the sides. It dissolves adhesives. It makes me say bad words in multiple languages. Bigger drops + no oil = fewer bad words. Lisa -- Lisa A. Nagy, Ph.D. University of Alabama-Birmingham [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Patrick Shaw Stewart Sent: Friday, January 18, 2008 2:20 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fwd: [ccp4bb] crystallisation robot One thing that people often overlook is that quite a lot of protein can be lost by denaturation on the surface of the drop. This is more significant for smaller drops. Two suggestions: (1) increase the proportion of protein in the - technical term - teeny drop to say two thirds and (2) cover the drops with oil eg Al's oils (silicone/paraffin). You still get vapor diffusion though the oil , and you'd like to slow up equilibration. of course (2) slows up the robotics a little, but both should be trivial to set up..
Re: [ccp4bb] crystallisation robot
Hi Joe, We have a 32-head Honeybee robot which sets up a 96-well plate with a single drop per well in ~6 minutes and 3 drops per well at ~9 minutes. A 96-head phoenix or hummingbird-like system is likely going to be faster, but not by that much. Our robot has its own humidified cabinet and as long as you keep evaporation under control I don't think set-up speed, within reason, is that important. Bart JOE CRYSTAL wrote: Hi, Does anyone have information about how long it takes to set up a 96-well tray for the crystallization robots available? Besides cost per tray and maintenance cost, another important feature we consider is the time for setting up a 96-well tray. It is an important factor since we are talking about sub-microliter drops. Best, Joe On Fri, Jan 18, 2008 at 12:28 PM, Lisa A Nagy [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] wrote: Al's Oil on the plates: What a nightmare!!! The oil creeps up the plate and over the sides. It dissolves adhesives. It makes me say bad words in multiple languages. Bigger drops + no oil = fewer bad words. Lisa -- Lisa A. Nagy, Ph.D. University of Alabama-Birmingham [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Patrick Shaw Stewart Sent: Friday, January 18, 2008 2:20 AM To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fwd: [ccp4bb] crystallisation robot One thing that people often overlook is that quite a lot of protein can be lost by denaturation on the surface of the drop. This is more significant for smaller drops. Two suggestions: (1) increase the proportion of protein in the - technical term - teeny drop to say two thirds and (2) cover the drops with oil eg Al's oils (silicone/paraffin). You still get vapor diffusion though the oil , and you'd like to slow up equilibration. of course (2) slows up the robotics a little, but both should be trivial to set up.. -- == Bart Hazes (Assistant Professor) Dept. of Medical Microbiology Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax:1-780-492-7521 ==
Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot
About 2 - 2.5 minutes on Mosquito for single-drop protocol, scaled according to the number of drops per well (7 minutes for 3-drop trays). About 25 minutes on a Tecan-derived platform (numbers vary greatly depending on the particular configuration). Artem _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of JOE CRYSTAL Sent: Monday, April 14, 2008 5:10 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot Hi, Does anyone have information about how long it takes to set up a 96-well tray for the crystallization robots available? Besides cost per tray and maintenance cost, another important feature we consider is the time for setting up a 96-well tray. It is an important factor since we are talking about sub-microliter drops. Best, Joe On Fri, Jan 18, 2008 at 12:28 PM, Lisa A Nagy [EMAIL PROTECTED] wrote: Al's Oil on the plates: What a nightmare!!! The oil creeps up the plate and over the sides. It dissolves adhesives. It makes me say bad words in multiple languages. Bigger drops + no oil = fewer bad words. Lisa -- Lisa A. Nagy, Ph.D. University of Alabama-Birmingham [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Patrick Shaw Stewart Sent: Friday, January 18, 2008 2:20 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fwd: [ccp4bb] crystallisation robot One thing that people often overlook is that quite a lot of protein can be lost by denaturation on the surface of the drop. This is more significant for smaller drops. Two suggestions: (1) increase the proportion of protein in the - technical term - teeny drop to say two thirds and (2) cover the drops with oil eg Al's oils (silicone/paraffin). You still get vapor diffusion though the oil , and you'd like to slow up equilibration. of course (2) slows up the robotics a little, but both should be trivial to set up..
[ccp4bb] Fwd: [ccp4bb] crystallisation robot
One thing that people often overlook is that quite a lot of protein can be lost by denaturation on the surface of the drop. This is more significant for smaller drops. Two suggestions: (1) increase the proportion of protein in the - technical term - teeny drop to say two thirds and (2) cover the drops with oil eg Al's oils (silicone/paraffin). You still get vapor diffusion though the oil , and you'd like to slow up equilibration. of course (2) slows up the robotics a little, but both should be trivial to set up.. On 1/17/08, Oganesyan, Vaheh [EMAIL PROTECTED] wrote: Mark, What was the state of the larger drops when tiny counterparts had crystals? My guess - they all precipitated. I'm trying to understand why some proteins or some conditions require change in protein concentration while others do not when migrating from smaller drops to larger ones. If it is protein dependent then I'm afraid there might be no one answer; if it is not then there should be a trend and explanation of phenomena. Vaheh From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Wednesday, January 16, 2008 8:31 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallisation robot Once upon a time I worked in a group that was interested in developing crystallization in microfluidics. This was before the time that Fluidigm existed and we had not heard of crystallization with the aid of microfluidics at the time. We had good reason to try to make a system that was as small and light as possible - it had something to do with the cost of shipping proteins and precipitants - less was better. And we also wanted all protein drops to be fully enclosed, out of safety considerations. Like Tassos, we were very worried what would happen if you scaled back drops along the lines of this discussion - several uL downto tens of nanoliters. If the stochastic process had a major influence over this process, we thought that we would never get any crystals. So we set up side-by-side experiments at larger volumes and smaller volumes - basically scanning several orders of magnitude - expecting a decrease of the number of crystals when volumes decrease. To our great surprise the outcome was that smaller volumes almost always gave MORE (I almost want to say 'dramatically more') crystals, more nucleation, and indeed in various cases the crystals grew much faster also. Indeed, it was trivial to observe that the surface-to-volume ratio was the primary driver for the nucleation process. We had control over geometry to some extent and were able to observe surfaces while crystals grow. The crystals would most commonly nucleate on a surface. So although there probably is something to stochastic aspects, it is clear that other aspects can be more important and overrule the stochastic considerations. The somewhat unpleasant consquence is of course that results acquired in very small volumes (with larger surface-to-volume ratio) cannot necessarily be repeated in larger volumes (smaller surface-to-volume ratio). This is not a flame, even if heat might be a good thing on a night with temperatures predicted far below 0F. :-) Mark -Original Message- From: Anastassis Perrakis [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 16 Jan 2008 6:17 am Subject: Re: [ccp4bb] crystallisation robot Oryxnano 50+50 nL Demetres Which, indirectly, brings up an interesting (but not relevant to the Oryx) question. Nucleation is a process that does have a stochastic aspect. Thus, one could argue that compromising to 200-300 nl might be better than either extremes of 50nl (too small volume and less chance for nucleation) or 1000 nl (too much sample). any comments ? (let the flames begin). A. PS1 another interesting issue that has has been hardly touched in these emails is the real sample loss: left in wells and not easy to recover, lost because of contamination with system liquid, etc ... PS2 I see lots of people with new robots. please do have a look at the www.BIOXHIT.org page and if you have a few minutes to assemble a table we will be happy to add your specs to our pages. it can be a nice resource and it has already enough things and already one response to my last email ;-) To make life easier to potential contributors we can provide an Excel sheet to fill up with your specs - just ask. On Jan 16, 2008, at 12:46, Demetres D. Leonidas wrote: David Briggs wrote: I'll defend the honour of the phoenix... (again) Bernhard Rupp 100+100 nl Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl Others.. Only time we have ANY problems is when the nano dispensing tip gets clogged. Often a good wash whilst still on the machine will clear the blockage. Dave -- David C
Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot
Al's Oil on the plates: What a nightmare!!! The oil creeps up the plate and over the sides. It dissolves adhesives. It makes me say bad words in multiple languages. Bigger drops + no oil = fewer bad words. Lisa -- Lisa A. Nagy, Ph.D. University of Alabama-Birmingham [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Patrick Shaw Stewart Sent: Friday, January 18, 2008 2:20 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fwd: [ccp4bb] crystallisation robot One thing that people often overlook is that quite a lot of protein can be lost by denaturation on the surface of the drop. This is more significant for smaller drops. Two suggestions: (1) increase the proportion of protein in the - technical term - teeny drop to say two thirds and (2) cover the drops with oil eg Al's oils (silicone/paraffin). You still get vapor diffusion though the oil , and you'd like to slow up equilibration. of course (2) slows up the robotics a little, but both should be trivial to set up..
Re: [ccp4bb] crystallisation robot
This illustrates nicely that the number of crystals per volume unit ultimately depends on the number of nucleants per volume unit. If the surface of your vessel has the power to nucleate it dramatically changes the effect of going to smaller volumes. I guess everybody is right here, depending on the vessel they use. Flip [EMAIL PROTECTED] schreef: Once upon a time I worked in a group that was interested in developing crystallization in microfluidics. This was before the time that Fluidigm existed and we had not heard of crystallization with the aid of microfluidics at the time. We had good reason to try to make a system that was as small and light as possible - it had something to do with the cost of shipping proteins and precipitants - less was better. And we also wanted all protein drops to be fully enclosed, out of safety considerations. Like Tassos, we were very worried what would happen if you scaled back drops along the lines of this discussion - several uL downto tens of nanoliters. If the stochastic process had a major influence over this process, we thought that we would never get any crystals. So we set up side-by-side experiments at larger volumes and smaller volumes - basically scanning several orders of magnitude - expecting a decrease of the number of crystals when volumes decrease. To our great surprise the outcome was that smaller volumes almost always gave MORE (I almost want to say 'dramatically more') crystals, more nucleation, and indeed in various cases the crystals grew much faster also. Indeed, it was trivial to observe that the surface-to-volume ratio was the primary driver for the nucleation process. We had control over geometry to some extent and were able to observe surfaces while crystals grow. The crystals would most commonly nucleate on a surface. So although there probably is something to stochastic aspects, it is clear that other aspects can be more important and overrule the stochastic considerations. The somewhat unpleasant consquence is of course that results acquired in very small volumes (with larger surface-to-volume ratio) cannot necessarily be repeated in larger volumes (smaller surface-to-volume ratio). This is not a flame, even if heat might be a good thing on a night with temperatures predicted far below 0F. ?:-) Mark -Original Message- From: Anastassis Perrakis [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 16 Jan 2008 6:17 am Subject: Re: [ccp4bb] crystallisation robot Oryxnano 50+50 nL? ? Demetres? ? ? Which, indirectly, brings up an interesting (but not relevant to the Oryx) question.? ? Nucleation is a process that does have a stochastic aspect.? ? Thus, one could argue that compromising to 200-300 nl might be better than either extremes of 50nl (too small volume and less chance for nucleation) or 1000 nl (too much sample).? ? any comments ? (let the flames begin).? ? A.? ? PS1? another interesting issue that has has been hardly touched in these emails is the real sample loss: left in wells and not easy to recover, lost because of contamination with system liquid, etc ...? ? PS2? I see lots of people with new robots. please do have a look at the www.BIOXHIT.org page and if you have a few minutes to assemble a table we will be happy to add your specs to our pages. it can be a nice resource and it has already enough things and already one response to my last email ;-) To make life easier to potential contributors we can provide an Excel sheet to fill up with your specs - just ask.? ? On Jan 16, 2008, at 12:46, Demetres D. Leonidas wrote:? ? ? David Briggs wrote:? I'll defend the honour of the phoenix... (again)? ? Bernhard Rupp 100+100 nl? Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl? Others..? ? Only time we have ANY problems is when the nano dispensing tip gets clogged. Often a good wash whilst still on the machine will clear the blockage.? ? Dave? ? ? ? ? -- ? David C. Briggs PhD? Father Crystallographer? http://www.dbriggs.talktalk.net http://www.dbriggs.talktalk.net? AIM ID: dbassophile? ? ? -- Demetres D. Leonidas, Ph.D.? Structural Biology Chemistry Group? Institute of Organic and Pharmaceutical Chemistry? The National Hellenic Research Foundation? 48, Vassileos Constantinou Avenue? Athens 116 35, Greece? ==? Tel. +30 210 7273841 (office)? +30 210 7273895 (lab) Fax. +30 210 7273831? E-mail: [EMAIL PROTECTED] URL: http://athena.eie.gr? ==? More new features than ever. Check out the new AIM(R) Mail ! - http://webmail.aim.com --- Flip Hoedemaeker Ph.D, CEO Key Drug Prototyping BV
Re: [ccp4bb] crystallisation robot
Mark, What was the state of the larger drops when tiny counterparts had crystals? My guess - they all precipitated. I'm trying to understand why some proteins or some conditions require change in protein concentration while others do not when migrating from smaller drops to larger ones. If it is protein dependent then I'm afraid there might be no one answer; if it is not then there should be a trend and explanation of phenomena. Vaheh From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Wednesday, January 16, 2008 8:31 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallisation robot Once upon a time I worked in a group that was interested in developing crystallization in microfluidics. This was before the time that Fluidigm existed and we had not heard of crystallization with the aid of microfluidics at the time. We had good reason to try to make a system that was as small and light as possible - it had something to do with the cost of shipping proteins and precipitants - less was better. And we also wanted all protein drops to be fully enclosed, out of safety considerations. Like Tassos, we were very worried what would happen if you scaled back drops along the lines of this discussion - several uL downto tens of nanoliters. If the stochastic process had a major influence over this process, we thought that we would never get any crystals. So we set up side-by-side experiments at larger volumes and smaller volumes - basically scanning several orders of magnitude - expecting a decrease of the number of crystals when volumes decrease. To our great surprise the outcome was that smaller volumes almost always gave MORE (I almost want to say 'dramatically more') crystals, more nucleation, and indeed in various cases the crystals grew much faster also. Indeed, it was trivial to observe that the surface-to-volume ratio was the primary driver for the nucleation process. We had control over geometry to some extent and were able to observe surfaces while crystals grow. The crystals would most commonly nucleate on a surface. So although there probably is something to stochastic aspects, it is clear that other aspects can be more important and overrule the stochastic considerations. The somewhat unpleasant consquence is of course that results acquired in very small volumes (with larger surface-to-volume ratio) cannot necessarily be repeated in larger volumes (smaller surface-to-volume ratio). This is not a flame, even if heat might be a good thing on a night with temperatures predicted far below 0F. :-) Mark -Original Message- From: Anastassis Perrakis [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 16 Jan 2008 6:17 am Subject: Re: [ccp4bb] crystallisation robot Oryxnano 50+50 nL Demetres Which, indirectly, brings up an interesting (but not relevant to the Oryx) question. Nucleation is a process that does have a stochastic aspect. Thus, one could argue that compromising to 200-300 nl might be better than either extremes of 50nl (too small volume and less chance for nucleation) or 1000 nl (too much sample). any comments ? (let the flames begin). A. PS1 another interesting issue that has has been hardly touched in these emails is the real sample loss: left in wells and not easy to recover, lost because of contamination with system liquid, etc ... PS2 I see lots of people with new robots. please do have a look at the www.BIOXHIT.org page and if you have a few minutes to assemble a table we will be happy to add your specs to our pages. it can be a nice resource and it has already enough things and already one response to my last email ;-) To make life easier to potential contributors we can provide an Excel sheet to fill up with your specs - just ask. On Jan 16, 2008, at 12:46, Demetres D. Leonidas wrote: David Briggs wrote: I'll defend the honour of the phoenix... (again) Bernhard Rupp 100+100 nl Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl Others.. Only time we have ANY problems is when the nano dispensing tip gets clogged. Often a good wash whilst still on the machine will clear the blockage. Dave -- David C. Briggs PhD Father Crystallographer http://www.dbriggs.talktalk.net http://www.dbriggs.talktalk.net AIM ID: dbassophile -- Demetres D. Leonidas, Ph.D. Structural Biology Chemistry Group Institute of Organic and Pharmaceutical Chemistry The National Hellenic Research Foundation 48, Vassileos Constantinou Avenue Athens 116 35, Greece == Tel. +30 210 7273841 (office) +30 210 7273895 (lab) Fax. +30 210 7273831 E-mail: [EMAIL PROTECTED] URL: http://athena.eie.gr == size=2 width=100
Re: [ccp4bb] crystallisation robot
Vaheh, I don't recall precipitation at all, but I do remember that we were prepared to change the crystallization recipes (i.e. adjust the recipes from previous 'large volume' crystallization to make more nuclei). For example, our first tests were with lysozyme (sorry, hardly a representative protein, I know) and it is known (paper by RA Judge et al) that buffer pH is a major determinant in nucleation (for that protein). Our initial concern was: is it possible to grow crystals in exceedingly small volumes? So we changed the pH to increase the nucleation rate. I think I recall that in case of lysozyme the nucleation rate is roughly inversely proportional to acid concentration, i.e. increase pH by 1 unit (10X less acid), gives ~10 times more nuclei. In general (for other proteins) we did make some minor adjustments in crystallization, but generally only in protein/precipitant concentrations. 'Minor' means adjustments of about 10% or so - going from 10% precipitant to 9% or 11%. When you go to very small volumes there is another consideration that you have to think about: mixing. If you assume that mixing takes place by diffusion (no stirring, it is very difficult to stir a very small volume reliably, i.e. to the same extent every time, unless you use microfluidic flow, in which case complete mixing to homogeneous mixtures is trivial - note: the Fluidigm system is 'microstatic', not microfluidic), so - if you assume mixing to take place by diffusion only, then the diffusion length in 100nL drops is very small and you can calculate in the worst case scenario how long it will take to accomplish full mixing by diffusion only. The time scale will be in the order of seconds. If you do the same calculation for a large drop (1 uL), the time scale is MUCH longer. So in case of batch crystallization, the 'end point' is reached very quickly in small volumes, while it takes much longer in large drops. If the end point causes protein precipitation, then this will happen very quickly after starting the experiment. In the larger scale experiment it will take much longer to reach precipitation and your system may go slowly through a process of enabling crystal growth before you reach drastic insolubility that causes precipitation. So as someone else said, the kinetics are completely different, the end point is not, and this can significantly affect the outcome of the experiment. Mark -Original Message- From: Oganesyan, Vaheh [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Thu, 17 Jan 2008 7:40 am Subject: Re: [ccp4bb] crystallisation robot Mark, What was the state of the larger drops when tiny counterparts had crystals? My guess - they all precipitated. I’m trying to understand why some proteins or some conditions require change in protein concentration while others do not when migrating from smaller drops to larger ones. If it is protein dependent then I’m afraid there might be no one answer; if it is not then there should be a trend and explanation of phenomena. Vaheh From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Wednesday, January 16, 2008 8:31 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallisation robot Once upon a time I worked in a group that was interested in developing crystallization in microfluidics. This was before the time that Fluidigm existed and we had not heard of crystallization with the aid of microfluidics at the time. We had good reason to try to make a system that was as small and light as possible - it had something to do with the cost of shipping proteins and precipitants - less was better. And we also wanted all protein drops to be fully enclosed, out of safety considerations. Like Tassos, we were very worried what would happen if you scaled back drops along the lines of this discussion - several uL downto tens of nanoliters. If the stochastic process had a major influence over this process, we thought that we would never get any crystals. So we set up side-by-side experiments at larger volumes and smaller volumes - basically scanning several orders of magnitude - expecting a decrease of the number of crystals when volumes decrease. To our great surprise the outcome was that smaller volumes almost always gave MORE (I almost want to say 'dramatically more') crystals, more nucleation, and indeed in various cases the crystals grew much faster also. Indeed, it was trivial to observe that the surface-to-volume ratio was the primary driver for the nucleation process. We had control over geometry to some extent and were able to observe surfaces while crystals grow. The crystals would most commonly nucleate on a surface. So although there probably is something to stochastic aspects, it is clear that other aspects can be more important and overrule the stochastic considerations. The somewhat
Re: [ccp4bb] crystallisation robot
I'll defend the honour of the phoenix... (again) Bernhard Rupp 100+100 nl Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl Others.. Only time we have ANY problems is when the nano dispensing tip gets clogged. Often a good wash whilst still on the machine will clear the blockage. Dave -- David C. Briggs PhD Father Crystallographer http://www.dbriggs.talktalk.net AIM ID: dbassophile
Re: [ccp4bb] crystallisation robot
Dear Bernard, I do not have long experience using Phoenix. I tried the machine during a couple of demonstrations I had chance to participate and also learned about other people experience who used the robot (various MPIs and universities in Germany and a couple of users from industry in Europe), I hope they can comment further. The crash of Phoenix needles that dissolved a myth on non-breakable Phoenix needles we witnessed during Art Robbins instruments presentation in PSDI meeting, 28th - 30th October 2007 Autrans, France. The needles were bended by rep during demonstration in the first day of the conference so that the robot head was moving without needles for the rest of the conference, it was funny. For the maintenance, all labs I know those using Phoenix have someone looking after the machine. In some lab setups the situation exaggerated even further: lab members do not have direct access to the delicate machine and all the crystallization by Phoenix is carried out by person who is trained as the User and dedicated to. As being quite experienced user of Cartesian nano dispenser (1 year), I can understand the problems of cleanness for the robots with not dispensable and therefore requiring washing steps needles/tips. As I know, the situation is significantly improved in Innovadyne screenmaker, for those who prefer flexibilities of 96 channel system over speed, simplicity, no cross contamination, accuracy of pipetting high PEGs Mosquito system (4 years experience). The electrostatic effect I mentioned was observed on the plates performed by Phoenix, while setting the same plates (Greiner triple, Corning 3550) by Mosquito did not cause similar problems (same drops volumes, same screening solutions), I hope colleagues may detail further. I would also suggest experienced Phoenix users to let us know what drops volume is routinely set in their labs. Bernhard Rupp 100+100 nl Others??? Others.. All the best, Alexey. -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Bernhard Rupp Sent: Wednesday, January 16, 2008 1:53 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallisation robot Dear Alexey, being involved in the development of the 'fixed needles + a few' robots and 96-well plates early on, I wonder about your bad experiences. You seem to say that the Phoenix requires more maintenance than the Mosquito - how long have you had that Phoenix or what model is giving you the trouble? For the electrostatics issue: it’s the plates that are electrostatic not the robot. This is a common lament for plastic plate users, and the Intelliplate designed to go with the Phoenix comes in anti-static bags as used in electronic component packaging. Other plates have anti-static coating, and there may be other tricks as well to avoid electrostatic charge (comments anyone?) Also, the limitation of the 200+200 you claim for the Phoenix seems extreme, users have set up 200+200 already with the old hydra+1 contraptions, and 100+100 seems to work reliable with the Phoenix (maybe some users can comment?). Could it be that yours is not set up correctly? I am sure ARI will be happy to send a European rep by to check. Best regards, BR -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Alexey Rak Sent: Monday, January 14, 2008 9:36 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallisation robot we have recently tried the new lid mentioned below. It works very well, Mosquito makes 3 hanging drops 96 well plate in 3-4 minutes! The film is very easy to handle and it is very transparent. The price for the lid is significantly reduced: from 13€ before to 4€ now. Comparing to Cartesian and Phoenix robots the Mosquito is REALLY maintenance free machine, especially the third generation insect where many 1st and 2nd Mosquito generation users advices have been implicated. Alexey Rak Structural Biology, Chemical Sciences Sanofi-Aventis Centre de Recherche Paris
Re: [ccp4bb] crystallisation robot
Oryxnano 50+50 nL Demetres David Briggs wrote: I'll defend the honour of the phoenix... (again) Bernhard Rupp 100+100 nl Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl Others.. Only time we have ANY problems is when the nano dispensing tip gets clogged. Often a good wash whilst still on the machine will clear the blockage. Dave -- David C. Briggs PhD Father Crystallographer http://www.dbriggs.talktalk.net http://www.dbriggs.talktalk.net AIM ID: dbassophile -- Demetres D. Leonidas, Ph.D. Structural Biology Chemistry Group Institute of Organic and Pharmaceutical Chemistry The National Hellenic Research Foundation 48, Vassileos Constantinou Avenue Athens 116 35, Greece == Tel. +30 210 7273841 (office) +30 210 7273895 (lab) Fax. +30 210 7273831 E-mail: [EMAIL PROTECTED] URL: http://athena.eie.gr ==
Re: [ccp4bb] crystallisation robot
In our original work on a prototype, we used the Cartesian technology. We were able to dispense 10nL+10nL drops with a large range of viscosities without difficulty. The main issue was to wash the tips after use to prevent clogging. That was with a system that could dispense 1nL droplets. The present systems are sub-nL now. We didn't have a problem with electrostatics. More recently, I've looked at all of the crystallization robot vendors. For single lab users, all of the systems work well. Systems like the Hydra or Mosquito are less automatic, but provide the basic functions for crystallization trial setup. For more of a user facility with a large number of users from several groups, you want more automation to avoid protocols that can damage the components, like alignment or breakage of the needles. You also want to consider the total annual cost of expendables and maintenance. Bernie Santarsiero On Wed, January 16, 2008 5:46 am, Demetres D. Leonidas wrote: Oryxnano 50+50 nL Demetres David Briggs wrote: I'll defend the honour of the phoenix... (again) Bernhard Rupp 100+100 nl Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl Others.. Only time we have ANY problems is when the nano dispensing tip gets clogged. Often a good wash whilst still on the machine will clear the blockage. Dave -- David C. Briggs PhD Father Crystallographer http://www.dbriggs.talktalk.net http://www.dbriggs.talktalk.net AIM ID: dbassophile -- Demetres D. Leonidas, Ph.D. Structural Biology Chemistry Group Institute of Organic and Pharmaceutical Chemistry The National Hellenic Research Foundation 48, Vassileos Constantinou Avenue Athens 116 35, Greece == Tel. +30 210 7273841 (office) +30 210 7273895 (lab) Fax. +30 210 7273831 E-mail: [EMAIL PROTECTED] URL: http://athena.eie.gr ==
Re: [ccp4bb] crystallisation robot
More recently, I've looked at all of the crystallization robot vendors. For single lab users, all of the systems work well. Systems like the Hydra or Mosquito are less automatic, but provide the basic functions for crystallization trial setup. For more of a user facility with a large number of users from several groups, you want more automation to avoid protocols that can damage the components, like alignment or breakage of the needles. You also want to consider the total annual cost of expendables and maintenance. There are two major kinds of a user facility with a large number of users from several groups a. One with an operator: then buy what the operator likes ;-) basically all machines do work b. NO operator: buy something that is hard to break and needs minimal maintenance We are in b. and happy with our Mosquito for 4 years or more now, although I am sure the other choices mentioned are also clearly fine. A.
Re: [ccp4bb] crystallisation robot - volume?
Regarding stochastic processes: to increase the chances of nucleation
one would like to have protein in the right nucleation zone for a
certain period of time.
If the drop becomes too small, the ratio evaporation surface/drop
volume changes. So, the nucleation zone might be reached within the
drop but either too fast ('racing through the phase diagram too
quickly') or in the smaller droplet turbulence caused by evaporation
might be more pronounced and interfere with nucleation.
At some stage I looked at this myself, although not for tiny volumes
(0.3, 0.5 and 0.8ul droplets). With smaller volume I saw more and
faster nucleation, but less crystal growth. There's got to be an
optimum in terms of drop volume when using vapour diffusion. Any
comments?
Carien
On 16 Jan 2008, at 13:17, Anastassis Perrakis wrote:
Oryxnano 50+50 nL
Demetres
Which, indirectly, brings up an interesting (but not relevant to the
Oryx) question.
Nucleation is a process that does have a stochastic aspect.
Thus, one could argue that compromising to 200-300 nl might be better
than either extremes of 50nl (too small volume and less chance for
nucleation) or 1000 nl (too much sample).
any comments ? (let the flames begin).
A.
PS1
another interesting issue that has has been hardly touched in these
emails is the real sample loss: left in wells and not easy to recover,
lost because of contamination with system liquid, etc ...
PS2
I see lots of people with new robots. please do have a look at the
www.BIOXHIT.org page and if you have a few minutes to assemble a table
we will be happy to add your specs to our pages. it can be a nice
resource and it has already enough things and already one response to
my last email ;-) To make life easier to potential contributors we can
provide an Excel sheet to fill up with your specs - just ask.
On Jan 16, 2008, at 12:46, Demetres D. Leonidas wrote:
David Briggs wrote:
I'll defend the honour of the phoenix... (again)
Bernhard Rupp 100+100 nl
Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl
Others..
Only time we have ANY problems is when the nano dispensing tip gets
clogged. Often a good wash whilst still on the machine will clear
the blockage.
Dave
--
David C. Briggs PhD
Father Crystallographer
http://www.dbriggs.talktalk.net http://www.dbriggs.talktalk.net
AIM ID: dbassophile
--
Demetres D. Leonidas, Ph.D.
Structural Biology Chemistry Group
Institute of Organic and Pharmaceutical Chemistry
The National Hellenic Research Foundation
48, Vassileos Constantinou Avenue
Athens 116 35, Greece
==
Tel. +30 210 7273841 (office)
+30 210 7273895 (lab) Fax. +30 210 7273831
E-mail: [EMAIL PROTECTED]
URL: http://athena.eie.gr
==
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Re: [ccp4bb] crystallisation robot
I have been using Phoenix for more than two years and so far there were no issues with maintenance. Wash the needles and nano-dispenser before and after the runs and you are good to go. Electrostatic effects have been seen in a way of drops being positioned on the side of the flat bottom plate, but the drops do not climb too high in my case probably because I'm using fairly large drops (200 + 200 nl). No matter how robust the system is, there will be always a way to break it. Once in a while I get large crystal that can be used for data collection, but in most of the cases optimization in hanging 1+1 uL drops is required. What I found quite difficult to do is to choose the appropriate protein concentration when moving from 200+200nL to 1+1 uL. Sometimes protein should be diluted 3-4 times, sometimes it shouldn't. How others are approaching this issue? Vaheh -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Anastassis Perrakis Sent: Wednesday, January 16, 2008 8:23 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallisation robot More recently, I've looked at all of the crystallization robot vendors. For single lab users, all of the systems work well. Systems like the Hydra or Mosquito are less automatic, but provide the basic functions for crystallization trial setup. For more of a user facility with a large number of users from several groups, you want more automation to avoid protocols that can damage the components, like alignment or breakage of the needles. You also want to consider the total annual cost of expendables and maintenance. There are two major kinds of a user facility with a large number of users from several groups a. One with an operator: then buy what the operator likes ;-) basically all machines do work b. NO operator: buy something that is hard to break and needs minimal maintenance We are in b. and happy with our Mosquito for 4 years or more now, although I am sure the other choices mentioned are also clearly fine. A.
Re: [ccp4bb] crystallisation robot
Vaheh, If I'm optimising in 24 well trays after a hit in 96 I generally use sitting drop trays (cryschem) as a first step and try different ratios of protein:mother liquor first rather than changing the protein concentration. I know that sometimes the crystals stick to the plastic but I use the hypodermic trick mentioned on the BB a few days ago. On the subject of drop size - I was at the RAMC meeting where this was discussed and several attendees had looked at the issue, 300+300nl or 400+400nl were mentioned as more optimal drop sizes for obtaining crystal hits during screening.See summary on: http://www.hamptonresearch.com/stuff/RAMC/RAMC2007Notes.aspx At York we use 150+150nl routinely for screening using the mosquito. I've moved up to 200+200 since the conference! Shirley Roberts ps I think I was responsible for the z to s change in the title begin:vcard fn:Shirley M. Roberts n:Roberts;Shirley M. org:York Structural Biology Laboratory adr:University of York;;Chemistry Department;York;;YO10 5YW;UK email;internet:[EMAIL PROTECTED] tel;work:01904 328268 tel;fax:01904 328266 url:http://www.ysbl.york.ac.uk/people/5.htm version:2.1 end:vcard
Re: [ccp4bb] crystallisation robot
You optimize that variable as well in scale-up. That is the problem with nano-drop screening- sometimes it is near impossible to scale up the drops. You have to re-screen around the conditions (response surface methods can conserve experiments) because the kinetics change so much between nano and big drops. The smaller they are, the harder it is to scale up, so we compromise between protein consumption and scalability. I think with most of these machines, you _can_ tweak it to dispense in the 20-50nl range accurately. To do that, you have to avoid using an air gap- either by drawing (and diluting) your protein against a column of water, or by using some other liquid (oil) between your protein and the dispensing fluid. Many times these tiny drops yield crystals much faster because they have more surface area to volume. But once you have them- well- that's where I've pulled out my hair. Scale-up is hard, and for delicate proteins (1% crystalline hits), near impossible from the 20 nl drops. That's the main reason we use bigger drops. In the early stages of a project, there is often a great deal of batch to batch variability in the protein samples. Minimizing this, and getting consistent polydispersity measurements can go a long way toward making scale-ups easier. All this screening is for naught if you need to optimize using a second batch of protein which behaves differently from the first, or if your first/only batch is degraded by the time you have hits to optimize. If you are fortunate to be producing your own protein, you can personally make sure you are producing consistent batches. The worst thing you can do is screen on the hearts, then optimize/scaleup with the tails (of the protein peak in the purification). Personally, I think having a liquid handling robot is almost as important as a crystallization robot. Lisa P.S. Confidential to various sales people: 1. If you had visited us, you would have known we were going to buy another robot. 2. If you had visited us, you would have known I was in the group. 3. If a member of our group didn't contact you and request information for purchasing your fabulous robot based on your website/mailings/ads, then your marketing people (not us) have the problem. 4. Sole source justification. Features and Price. 20K is a significant difference, and we didn't need the extra features on your machine. Or we needed features yours didn't have. 5. Sending snarky email messages about me to my coworkers makes all of us less inclined to view your company favorably for future purchases. -- Lisa A. Nagy, Ph.D. University of Alabama-Birmingham [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Oganesyan, Vaheh Sent: Wednesday, January 16, 2008 9:06 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallisation robot ... Once in a while I get large crystal that can be used for data collection, but in most of the cases optimization in hanging 1+1 uL drops is required. What I found quite difficult to do is to choose the appropriate protein concentration when moving from 200+200nL to 1+1 uL. Sometimes protein should be diluted 3-4 times, sometimes it shouldn't. How others are approaching this issue? Vaheh
Re: [ccp4bb] crystallisation robot
Once upon a time I worked in a group that was interested in developing crystallization in microfluidics. This was before the time that Fluidigm existed and we had not heard of crystallization with the aid of microfluidics at the time. We had good reason to try to make a system that was as small and light as possible - it had something to do with the cost of shipping proteins and precipitants - less was better. And we also wanted all protein drops to be fully enclosed, out of safety considerations. Like Tassos, we were very worried what would happen if you scaled back drops along the lines of this discussion - several uL downto tens of nanoliters. If the stochastic process had a major influence over this process, we thought that we would never get any crystals. So we set up side-by-side experiments at larger volumes and smaller volumes - basically scanning several orders of magnitude - expecting a decrease of the number of crystals when volumes decrease. To our great surprise the outcome was that smaller volumes almost always gave MORE (I almost want to say 'dramatically more') crystals, more nucleation, and indeed in various cases the crystals grew much faster also. Indeed, it was trivial to observe that the surface-to-volume ratio was the primary driver for the nucleation process. We had control over geometry to some extent and were able to observe surfaces while crystals grow. The crystals would most commonly nucleate on a surface. So although there probably is something to stochastic aspects, it is clear that other aspects can be more important and overrule the stochastic considerations. The somewhat unpleasant consquence is of course that results acquired in very small volumes (with larger surface-to-volume ratio) cannot necessarily be repeated in larger volumes (smaller surface-to-volume ratio). This is not a flame, even if heat might be a good thing on a night with temperatures predicted far below 0F. ?:-) Mark -Original Message- From: Anastassis Perrakis [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 16 Jan 2008 6:17 am Subject: Re: [ccp4bb] crystallisation robot Oryxnano 50+50 nL? ? Demetres? ? ? Which, indirectly, brings up an interesting (but not relevant to the Oryx) question.? ? Nucleation is a process that does have a stochastic aspect.? ? Thus, one could argue that compromising to 200-300 nl might be better than either extremes of 50nl (too small volume and less chance for nucleation) or 1000 nl (too much sample).? ? any comments ? (let the flames begin).? ? A.? ? PS1? another interesting issue that has has been hardly touched in these emails is the real sample loss: left in wells and not easy to recover, lost because of contamination with system liquid, etc ...? ? PS2? I see lots of people with new robots. please do have a look at the www.BIOXHIT.org page and if you have a few minutes to assemble a table we will be happy to add your specs to our pages. it can be a nice resource and it has already enough things and already one response to my last email ;-) To make life easier to potential contributors we can provide an Excel sheet to fill up with your specs - just ask.? ? On Jan 16, 2008, at 12:46, Demetres D. Leonidas wrote:? ? ? David Briggs wrote:? I'll defend the honour of the phoenix... (again)? ? Bernhard Rupp 100+100 nl? Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl? Others..? ? Only time we have ANY problems is when the nano dispensing tip gets clogged. Often a good wash whilst still on the machine will clear the blockage.? ? Dave? ? ? ? ? -- ? David C. Briggs PhD? Father Crystallographer? http://www.dbriggs.talktalk.net http://www.dbriggs.talktalk.net? AIM ID: dbassophile? ? ? -- Demetres D. Leonidas, Ph.D.? Structural Biology Chemistry Group? Institute of Organic and Pharmaceutical Chemistry? The National Hellenic Research Foundation? 48, Vassileos Constantinou Avenue? Athens 116 35, Greece? ==? Tel. +30 210 7273841 (office)? +30 210 7273895 (lab) Fax. +30 210 7273831? E-mail: [EMAIL PROTECTED] URL: http://athena.eie.gr? ==? More new features than ever. Check out the new AIM(R) Mail ! - http://webmail.aim.com
Re: [ccp4bb] crystallisation robot
Once upon a time I worked in a group that was interested in developing crystallization in microfluidics. This was before the time that Fluidigm existed and we had not heard of crystallization with the aid of microfluidics at the time. We had good reason to try to make a system that was as small and light as possible - it had something to do with the cost of shipping proteins and precipitants - less was better. And we also wanted all protein drops to be fully enclosed, out of safety considerations. Like Tassos, we were very worried what would happen if you scaled back drops along the lines of this discussion - several uL downto tens of nanoliters. If the stochastic process had a major influence over this process, we thought that we would never get any crystals. So we set up side-by-side experiments at larger volumes and smaller volumes - basically scanning several orders of magnitude - expecting a decrease of the number of crystals when volumes decrease. To our great surprise the outcome was that smaller volumes almost always gave MORE (I almost want to say 'dramatically more') crystals, more nucleation, and indeed in various cases the crystals grew much faster also. Indeed, it was trivial to observe that the surface-to-volume ratio was the primary driver for the nucleation process. We had control over geometry to some extent and were able to observe surfaces while crystals grow. The crystals would most commonly nucleate on a surface. So although there probably is something to stochastic aspects, it is clear that other aspects can be more important and overrule the stochastic considerations. The somewhat unpleasant consquence is of course that results acquired in very small volumes (with larger surface-to-volume ratio) cannot necessarily be repeated in larger volumes (smaller surface-to-volume ratio). This is not a flame, even if heat might be a good thing on a night with temperatures predicted far below 0F. ?:-) Mark -Original Message- From: Anastassis Perrakis [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 16 Jan 2008 6:17 am Subject: Re: [ccp4bb] crystallisation robot Oryxnano 50+50 nL? ? Demetres? ? ? Which, indirectly, brings up an interesting (but not relevant to the Oryx) question.? ? Nucleation is a process that does have a stochastic aspect.? ? Thus, one could argue that compromising to 200-300 nl might be better than either extremes of 50nl (too small volume and less chance for nucleation) or 1000 nl (too much sample).? ? any comments ? (let the flames begin).? ? A.? ? PS1? another interesting issue that has has been hardly touched in these emails is the real sample loss: left in wells and not easy to recover, lost because of contamination with system liquid, etc ...? ? PS2? I see lots of people with new robots. please do have a look at the www.BIOXHIT.org page and if you have a few minutes to assemble a table we will be happy to add your specs to our pages. it can be a nice resource and it has already enough things and already one response to my last email ;-) To make life easier to potential contributors we can provide an Excel sheet to fill up with your specs - just ask.? ? On Jan 16, 2008, at 12:46, Demetres D. Leonidas wrote:? ? ? David Briggs wrote:? I'll defend the honour of the phoenix... (again)? ? Bernhard Rupp 100+100 nl? Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl? Others..? ? Only time we have ANY problems is when the nano dispensing tip gets clogged. Often a good wash whilst still on the machine will clear the blockage.? ? Dave? ? ? ? ? -- ? David C. Briggs PhD? Father Crystallographer? http://www.dbriggs.talktalk.net http://www.dbriggs.talktalk.net? AIM ID: dbassophile? ? ? -- Demetres D. Leonidas, Ph.D.? Structural Biology Chemistry Group? Institute of Organic and Pharmaceutical Chemistry? The National Hellenic Research Foundation? 48, Vassileos Constantinou Avenue? Athens 116 35, Greece? ==? Tel. +30 210 7273841 (office)? +30 210 7273895 (lab) Fax. +30 210 7273831? E-mail: [EMAIL PROTECTED] URL: http://athena.eie.gr? ==? More new features than ever. Check out the new AIM(R) Mail ! - http://webmail.aim.com
Re: [ccp4bb] crystallisation robot
Dear Alexey, being involved in the development of the 'fixed needles + a few' robots and 96-well plates early on, I wonder about your bad experiences. You seem to say that the Phoenix requires more maintenance than the Mosquito - how long have you had that Phoenix or what model is giving you the trouble? For the electrostatics issue: it’s the plates that are electrostatic not the robot. This is a common lament for plastic plate users, and the Intelliplate designed to go with the Phoenix comes in anti-static bags as used in electronic component packaging. Other plates have anti-static coating, and there may be other tricks as well to avoid electrostatic charge (comments anyone?) Also, the limitation of the 200+200 you claim for the Phoenix seems extreme, users have set up 200+200 already with the old hydra+1 contraptions, and 100+100 seems to work reliable with the Phoenix (maybe some users can comment?). Could it be that yours is not set up correctly? I am sure ARI will be happy to send a European rep by to check. Best regards, BR -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Alexey Rak Sent: Monday, January 14, 2008 9:36 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallisation robot we have recently tried the new lid mentioned below. It works very well, Mosquito makes 3 hanging drops 96 well plate in 3-4 minutes! The film is very easy to handle and it is very transparent. The price for the lid is significantly reduced: from 13€ before to 4€ now. Comparing to Cartesian and Phoenix robots the Mosquito is REALLY maintenance free machine, especially the third generation insect where many 1st and 2nd Mosquito generation users advices have been implicated. Alexey Rak Structural Biology, Chemical Sciences Sanofi-Aventis Centre de Recherche Paris
Re: [ccp4bb] crystallisation robot
we have recently tried the new lid mentioned below. It works very well, Mosquito makes 3 hanging drops 96 well plate in 3-4 minutes! The film is very easy to handle and it is very transparent. The price for the lid is significantly reduced: from 13€ before to 4€ now. Comparing to Cartesian and Phoenix robots the Mosquito is REALLY maintenance free machine, especially the third generation insect where many 1st and 2nd Mosquito generation users advices have been implicated. Alexey Rak Structural Biology, Chemical Sciences Sanofi-Aventis Centre de Recherche Paris -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Shirley Roberts Sent: Friday, January 11, 2008 7:25 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallisation robot I sent this personal reply on wednesday to Madhavi Nalam's original question. In the light of recent comments I'm posting it to the BB to support TTP labtech and Mosquito! Some of the points have been made by others now, excuse the repeats! We at YSBL have been very happy with our mosquito. We use it to setup sitting drops with the MRC Wilden plate, this can be for screening or optimisation (not always necessary to go to 24-well format). You can also setup hanging drops, this can work better if detergents are involved. A new lid has just been released from TTP that costs £2.50 and takes 3 drops per well but we havn't tried this yet. You will need another robot (or multichannel pipette) for transfering the screen solutions.We use the hydra from alpha-helix ltd with flexible needles so hard to break. The phoenix robot does both jobs and seems popular. The Douglas robot has been mentioned, the Cartesian is the other main robot in use but I don't have any personal experience of it, I've heard it is quite high maintenance. Good luck with your purchase! Shirley Roberts
Re: [ccp4bb] crystallisation robot
I sent this personal reply on wednesday to Madhavi Nalam's original question. In the light of recent comments I'm posting it to the BB to support TTP labtech and Mosquito! Some of the points have been made by others now, excuse the repeats! We at YSBL have been very happy with our mosquito. We use it to setup sitting drops with the MRC Wilden plate, this can be for screening or optimisation (not always necessary to go to 24-well format). You can also setup hanging drops, this can work better if detergents are involved. A new lid has just been released from TTP that costs £2.50 and takes 3 drops per well but we havn't tried this yet. You will need another robot (or multichannel pipette) for transfering the screen solutions.We use the hydra from alpha-helix ltd with flexible needles so hard to break. The phoenix robot does both jobs and seems popular. The Douglas robot has been mentioned, the Cartesian is the other main robot in use but I don't have any personal experience of it, I've heard it is quite high maintenance. Good luck with your purchase! Shirley Roberts begin:vcard fn:Shirley M. Roberts n:Roberts;Shirley M. org:York Structural Biology Laboratory adr:University of York;;Chemistry Department;York;;YO10 5YW;UK email;internet:[EMAIL PROTECTED] tel;work:01904 328268 tel;fax:01904 328266 url:http://www.ysbl.york.ac.uk/people/5.htm version:2.1 end:vcard
Re: [ccp4bb] crystallisation robot
Thanks to all who replied to my query. Madhavi
Re: [ccp4bb] crystallisation robot
Dear all - Under the BIOXHIT home page, http://www.bioxhit.org you can navigate to Section 1 or HTP Crystallization, or simply follow the link below: http://icarus.embl-hamburg.de/bioxhit/bioXHITSection1.jsp It has quite a lot of data on what people use, available facilities, and testing of some popular robots. If any lab/facility wants to contribute data to that resource please get in contact with me. My best regards, Tassos On 11 Jan 2008, at 19:25, Shirley Roberts wrote: I sent this personal reply on wednesday to Madhavi Nalam's original question. In the light of recent comments I'm posting it to the BB to support TTP labtech and Mosquito! Some of the points have been made by others now, excuse the repeats! We at YSBL have been very happy with our mosquito. We use it to setup sitting drops with the MRC Wilden plate, this can be for screening or optimisation (not always necessary to go to 24-well format). You can also setup hanging drops, this can work better if detergents are involved. A new lid has just been released from TTP that costs £2.50 and takes 3 drops per well but we havn't tried this yet. You will need another robot (or multichannel pipette) for transfering the screen solutions.We use the hydra from alpha-helix ltd with flexible needles so hard to break. The phoenix robot does both jobs and seems popular. The Douglas robot has been mentioned, the Cartesian is the other main robot in use but I don't have any personal experience of it, I've heard it is quite high maintenance. Good luck with your purchase! Shirley Robertsroberts.vcf
