Recursos Técnicos - Posters Científicos
Using BioTek’s Precision 2000 Automated Pipetting System for Microarray Sample Preparation
January 01, 2000
Authors: Paul Held, Applications Department, BioTek Instruments, Winooski, VT; Gavin Picket, Keck-UNM Genomics Resource, University of New Mexico, Albuquerque, NM
This poster was shown at International Symposium on Laboratory Automation and Robotics, October 14-17, 2001
The production of microarrays requires the spotting of large numbers of unique DNA fragments onto several different substrates. While several commercially available instruments have automated this spotting task, the sample preparation, culture propagation and maintenance of the DNA library are often performed manually with multichannel pipettes in 8 x 12, 96-well, or 16 x 24, 384-well formats. Manual multichannel pipettes, while more efficient than single-channel pipettes, still represent a large amount of pipetting with many opportunities for pipetting errors. Here we describe the use of the Precision 2000TM Automated Pipetting System to carry out many of the necessary pipetting tasks required for the preparation of samples for microarray spotting. These steps include: the propagation of plasmid libraries, PCR reaction preparation, treatment of PCR products for agarose gel electrophoresis, and the reconstitution of lyophilized samples prior to microarray spotting. The Precision 2000 has a completely configurable six-station platform to hold the required pipette tips, reagent troughs, and microplates (96- and 384-well) for fluid transfer. The platform is removable, allowing for multi-user friendliness, easy cleaning, and setup of the instrument. The 8-channel pipette arm moves up and down as well as side-to-side, while the platform moves front to back to provide complete access to all locations on the work platform and complete configurability. The pipette arm uses a proprietary technology to reliably pick up and seal any standard tip with individual, free-floating barrels that compensate for tips out of position. An optional rapid dispense 8-channel manifold, which uses a precise bi-directional syringe pump to accurately and rapidly dispense fluids from a large unpressurized reservoir, is also available. The Precision 2000 has a built-in microprocessor that controls all movements. The flexible software, both onboard and PC-based, provides complete programming capabilities. For more complete automation, robotics interfaces can be developed using ActiveX® software commands. The Precision 2000’s small size, with a 15 x 21-inch footprint and a height of 16 inches, allows it to be used almost anywhere including most biological safety cabinets or chemical fume hoods.
Figure 1: Precision 2000 Automated Pipetting System with dust cover.
- BioTek Precision 2000 Automated Microplate Pipetting System
- MJ Research Thermocycler
- Qiagen BioRobot 3000
- BioRobotics Microarray Spotter
- Savant Lyophilizer
- BioTek PowerWaveX Microplate Spectrophotometer
Materials and Methods
In order to determine the Precision 2000 low-volume pipetting capabilities, concentrated dye solutions were dispensed using the Precision 2000. In some experiments, blue dye solution in TRIS buffer diluent was dispensed into clean dry plates (either 96-well or 384-well) and colorless TRIS buffer diluent was added. The resulting absorbance was then read at 595 nm using a microplate absorbance reader. In other experiments, yellow dye was added to wells already containing the diluent. In these experiments, both the dye and the colorless buffer also contained 0.1% Tween 20 surfactant. After the addition of the aqueous solutions, the absorbance of each well of the plate was determined at 450 nm using a PowerWaveX absorbance reader (BioTek Instruments, Inc., Winooski, VT). The data from both experiments was then exported to Microsoft® Excel and the data reduction was performed.
Accuracy was determined using either a gravimetric method or the dilution of dye. Determinations using the gravimetric method were performed by weighing plates pre-filled with 100 μl of PBS using an analytical balance. After dispensing the appropriate fluid to the plate using the Precision 2000, the plate was quickly re-weighed. The resultant weight changes, when divided by the number of plate wells, returned an average per well dispense volume. When calculating the accuracy of dispense using the dye method, concentrated yellow dye was pipetted into pre-filled plates with the Precision 2000. After mixing on an orbital shaker for 60 seconds, the absorbance was read on a PowerWaveX microplate spectrophotometer (BioTek Instruments, Inc.). The resultant absorbance values were compared to a pre-existing calibration curve and the dispense volume interpolated.
The Precision 2000 Automated Pipetting system carries out many of the routine pipetting tasks associated with the sample preparation for microarray spotting. The Precision 2000 can be installed on any stable laboratory surface, including standard lab benches, as well as mobile carts. The small footprint even allows for use inside of a biosafety cabinet. The Precision 2000 can be programmed using Precision Powerä software running on an attached PC. After the programming has been completed, the necessary files are downloaded to the instrument and the Precision 2000 is capable of running stand-alone. These features provide the following advantages:
- The system can be quickly and easily moved to different locations and maintain reliable operation.
- The end user can reprogram the system as desired without the need of factory reinstallation or programming.
While there are numerous tasks that can be performed by the Precision 2000, here are descriptions of some of the tasks carried out by the Precision 2000. Note that grayed text indicate tasks in the process not carried out by the Precision 2000.
PCR Reaction Setup
Figure 2: Deck layout of Precision 2000 for yeast culture PCR setup.
The Precision 2000 transfers 80 µl of PCR master mix containing, polymerase enzyme, primers, and deoxynucleotides to all wells of a 96-well PCR plate (orange shading) from the reagent trough (yellow shading) using standard pipette tips (green shading). Using aerosol barrier tips (blue shading), the Precision 2000 transfers 20 µl of yeast culture in deep well plates to the 96-well PCR plate (orange shading).
- PCR plates are manually transferred to a MJ Research thermocycler where primer-specific thermocyling run is performed.
- Completed PCR reactions are then transferred to a Qiagen 3000 BioRobot for PCR product cleanup.
- After primer and nucleotide removal, PCR reactions are lyophilized to dryness with a Savant lyophilizer.
- Samples are then spotted using a BioRobotics Microarray spotting robot.
PCR Check-Gel Sample Preparation
Figure 3: Precision 2000 Deck Layout for Gel Sample Preparation.
Using aerosol tips (blue shading), the Precision 2000 transfers 4 μl of sample from the wells of the Qiagen collection plate to the corresponding wells of a round bottom plate. Using normal tips (green shading), 5 μl of gel loading buffer is added to the samples in the round bottom plate and mixed using the Precision 2000.
Figure 4: Close up picture of pipette arm with tips. Samples are being transferred froma 96-well into a 384-well microplate.
Results of Precision 2000 Low-Volume Pipetting
As demonstrated in Figure 5, when aqueous solutions were dispensed into dry plates, large well-to-well differences were observed with very small volumes (less than 3 μl). The Coefficient of Variance (%CV) for a plate with a 1 μl dispense was found to be as great as 60%. However, the well-to-well differences quickly diminish with increasing dispense volume. Dispense volumes as small as 3 μl have a %CV less than 4%. When small volumes of liquid are dispensed into 384-well plates, a similar pattern of well-to-well variation is observed (Figure 6). When dispensing into dry 384-well plates, very large differences between wells are observed at 1-2 μl. However, dispense volume greater than 3 μl demonstrate excellent results.
When small aqueous volumes are dispensed into partially filled plate wells, a marked improvement in the precision is observed (Figure 7.) Volumes as small as 1 μl can be dispensed with a coefficient of variance less than 4%. With dispense volumes of 3 μl per well or greater, the %CV is routinely less than 2%. The pattern of increased precision with dispense volumes of 3 μl or greater is similar in nature as observed when dispensing into dry wells. However, the degree of improvement is lessened as results of the vastly improved results at 1-2 μl dispense volumes (2-3.5% vs. 30-60 %CVs).
As demonstrated in Figure 8, the Precision 2000’s Rapid Dispense Mode option enables precise delivery of fluids as well. Using either a 96- or a 384-well plate, volumes as small as 10 μl can be dispensed from the bulk reagent dispense manifold. When dispensing 10 μl into a plate well, it appears that the 384-well plate offers better precision. However, at volumes of 20 μl l or greater, there appears to be little difference between the different plate matrices (Figure 8).
Figure 5: Dispense precision into dry 96-well plates using the Precision 2000 pipette at various dispense volumes. The indicated volume of TRIS buffer containing blue food coloring was dispensed to all the wells of a 96-well plate using the pipette. After dispensing the colored dye, 100 µl of TRIS buffer was dispensed into each well using the dispense manifold at a speed setting of 5. The absorbance at 595 nm for each well of every plate was determined using a microplate absorbance reader. Note that each data bar represents the %CV of an entire plate.
Figure 6: Dispense precision into dry 384-well plates using the Precision 2000 pipette at various dispense volumes. The indicated volume of TRIS buffer containing blue food coloring was dispensed to all the wells of a 384-well plate using the pipette. After dispensing the colored dye, 50 µl of TRIS buffer was dispensed into each well using the dispense manifold at a speed setting of 5. The absorbance at 595 nm for each well of every plate was determined using a microplate absorbance reader. Note that each data bar represents the %CV of an entire plate.
Figure 7: Dispense precision into partially filled 96-well plates using the Precision 2000 pipette at various dispense volumes. Into each well of a plate, 100 µl of QC check solution #3 was dispensed using the pipette as a diluent. Next, the indicated volume of yellow dye was dispensed to all the wells of a 96-well plate using the pipette. The absorbance at 450 nm for each well of every plate was determined using a PowerWaveX Microplate Spectrophotometer (BioTek Instruments, Winooski, VT). Note that each data bar represents the %CV of an entire plate.
Figure 8: Dispense precision into dry 96- or 384-well plates using the Precision 2000 manifold at various dispense volumes. The indicated volume of TRIS buffer containing blue food coloring was dispensed to all the wells of either a 96- or a 384-well plate using the manifold. After dispensing the colored dye, the buffer bottle was changed to TRIS buffer only and either 100 μl or 50 μl of TRIS buffer was dispensed into each well of the 96- or 384-well plates respectively using the manifold at a speed setting of 5. The absorbance at 595 nm for each well of every plate was determined using a microplate absorbance reader. Note that each data bar represents the %CV of an entire plate.
These data indicate that the Precision 2000 can be used to dispense the small aqueous volumes necessary for microarray sample preparation precisely and accurately. Use of the pipette arm allows for infinite flexibility in regards to fluid transfers. The manifold used in Precision 2000’s Rapid Dispense Mode option is also quite useful for low-volume dispenses. This bulk reagent dispense manifold has the advantage of being very rapid and is quite useful when dispensing down to as low as 10 µl per well.
Several factors influence the ability to dispense small volumes with the Precision 2000. The positive displacement syringes depend on the displacement of air to push the fluid out of the barrel of the tip. Unfortunately, gaseous materials, unlike liquids, are prone to compression, which would suggest that accuracy occurs at large volumes (e.g., 100 µl), where most of the column is liquid. This often precludes the ability to be accurate at distinctly smaller volumes. Because air is also prone to expansion and contraction as a result of temperature changes, having samples and reagents equilibrated to ambient temperature will result in more accurate and precise dispensing as well. For the most accurate and precise dispenses, it is advisable to include an initial pre-pickup of a small volume. This will provide some compensation for air gas compression, as well as provide a humid environment inside the pipette tip.
Dispensing into a liquid generally is more accurate and precise than dispensing into an empty well. This phenomenon is most likely due to small amount of the fluids wicking onto the outside of the pipette tip and not being dispensed into the well. When dispensing into a pre-existing liquid, that fluid is more likely to disperse into the fluid than onto the tip as a result of surface tension.
The Precision 2000 Automated Microplate Pipetting System is an accurate and precise liquid handling system capable of automating most routine pipetting, dispensing, and diluting applications. Ideal for a flexible and versatile automated system with its small footprint, it has proven to be very capable of low-volume pipetting (1 µl) to accommodate today’s low assay volumes.
The flexibility and mobility of this system also allows for many of the manual pipetting tasks required for microarray spotting to be automated.
Held, P. and Cohen, D. (2000) Using BioTek’s Precision 2000 to Dispense Small Volumes. Application Note, BioTek Instruments Inc., Highland Park, Box 998 Winooski, VT 05404, www.biotek.com
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