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Laboratory of Molecular Biophysics
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The Oxford LMB crystallisation facility currently has over 40 users and this number is growing due to the acquisition of new technologies designed to improve result throughput and scoring. In late 2002 the LMB purchased a TECAN liquid handling platform with the aim of fully automating the setup of macromolecular crystallisation trials. It was at first necessary to reconfigure the liquid handling system for crystallographic use. Since then the TECAN Genesis 150 has been operating effectively in its new role. We will briefly outline the set-up, level and mode of use. In October 2003, the LMB also acquired a high definition Leica microscope with an automated Prior stage in order to keep pace with the results produced by the robot.
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The reconfigured TECAN robot has a unique set-up that allows it to reproducibly pipette a variety of crystallisation screens. The liquid handling arm has a total of 8 tips: 4 standard volume tips, which pipette volumes between 250 µl and 0.5µl, and 4 low volume tips which pipette volumes between 1 and 0.25 µl. The standard tips are powered by 250 µl capacity syringes, and the low volume tips are powered by smaller 50 µl NPS (Nano-Pipetting System) syringes.
The original design of 50 µl syringes was identical to that of the larger 250 µl syringes: a glass chamber fitted with a Teflon capped aluminium syringe plunger. However, after several months of use, substantial wear and tear on the 50 µl syringes was noticed, which in turn significantly impaired the accuracy and reproducibility of low-volume pipetting. In consultation with TECAN the glass-clad aluminium plungers were replaced with more durable ceramic plungers enclosed in a plastic syringe body. These have been a great success and have certainly proved flexible enough to cope with the demands placed on the system.
Before leaving the LMB on his retirement Mike Pritchard was involved in the creation of a new rack for the robot, allowing samples to be pipetted directly from 48 x 1.5 ml micro tubes and kept on ice if necessary. This enables robot users to explore their crystallisation conditions further using various additive screens. Planned future developments for the robot include new anti-evaporation covers, and further programming to give users greater scope in setting up secondary crystallisation trials.
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The automated Leica microscope is now fully operational. Users of the microscope can either scan an entire 96 well crystallisation plate, or select an individual well for imaging. Picture quality and the speed of data processing are both highly satisfactory, with the system able to generate 96 high definition images in under 5 minutes. The lens depth of field is good enough to see the majority of crystalline particulates at the low magnifications required for plate scanning, but also performs excellently at the high magnifications required for more in-depth analysis. It has also been effective in overcoming problems of imaging at very low exposures necessary for viewing light-sensitive proteins. Planned future developments for the microscope include an image/crystallisation database for cataloguing interesting crystallisation characteristics. We are also continually testing new crystallisation plates for improved picture quality, although the images obtained with the current plates are highly satisfactory.
Other developments in the crystallisation facility include the operation of the Tecan SpectraFluor Plus, a fully automatic fluorimeter for measuring samples on a microplate. The Tecan robot is configured to pipette reagents on microplates, which can then be read on the fluorimeter. We also set up the S9 website which has proved a very useful tool for users of the facility.
The crystallisation facility boasts an ever-growing number of users, who are now profiting from the new technologies recently installed. The addition of the robot has increased not only throughput, but also output. The full impact of the microscope will become apparent once the crystallisation database is operational.
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