omation mbers - Society for Laboratory Automation and Screening

WP025
Duane Kubischta
DOE Joint Genome Institute
Instrumentation
2800 Mitchell Drive, B100
Walnut Creek, California 94598
DGKubischta@lbl.gov
Identifying and Reducing Crossover Contamination at the Sub-Microliter Level for Pipetting
Tips in a High Throughput DNA Sequencing Environment
In a high throughput sequencing environment, where hundreds of 384-well plates are processed daily, the cost
and loading efficiency associated with pipetting tips necessitates that the tips are used for processing multiple
plates. When these tips are re-used, despite various washing and cleaning steps, there is always a risk of crosscontamination.
This type of contamination was detected on a Beckman-Coulter Biomek FX, which is used for Solid
Phase Reversible Immobilization (SPRI) clean-up of labeled sequencing fragments. This paper will detail the detection
and characterization of this cross-contamination and efforts that were used to reduce its occurrence.At the U.S. DOE
Joint Genome Institute, a SPRI protocol is used with a magnetic bead, ethanol, and tetraethyleneglycol (BET) solution
to bind ssDNA in order to clean up the remaining cellular and sequencing chemistry debris from a Rolling Circle
Amplification (RCA) preparation process. This process is accomplished with a 384-well head on the Biomek FX. Plates
are processed in parallel batches of 8–10 and go through the following steps: BET addition, BET incubation, BET
removal on magnets, ethanol rinse on magnets, ethanol evaporation, and H20 resuspension and transfer. Pipetting
tips are currently washed with deionized H20. This work was performed under the auspices of the U.S. Department
of Energy’s Office of Science, Biological and Environmental Research Program and by the University of California,
Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48, Lawrence Berkeley National Laboratory
under contract No. DE-AC03-76SF00098 and Los Alamos National Laboratory under contract No. W-7405-ENG-36.
WP026
Scott Kuzdzal
PerkinElmerSCIEX
Proteomics
710 Bridgeport Avenue
Shelton, Connecticut 06484
scott.kuzdzal@perkinelmer.com
204
Co-Author(s)
Joe DiCesare, Mary Lopez,
Lisa Sapp, Tillmann Ziegert
Fully-Automated, High Throughput Peptide Mass Fingerprinting by MALDI Orthogonal-TOF
Mass Spectrometry
MALDI-TOF-MS has become an important analytical tool in the identification of proteins and evaluation of their
role in biological processes. While Peptide Mass Fingerprinting (PMF) provides accurate identification of proteins,
processing hundreds or thousands of samples a day can be labor and time intensive. The combination of
automated liquid handlers with orthogonal MALDI-TOF provides for extremely accurate, fully-automated analysis
of such samples. The prOTOF 2000 o-MALDI TOF supports 96 and 384 well stainless steel and disposable MALDI
targets. These sample targets are handled by an extremely precise high-speed source (accuracy of 2 microns).
Acquisition, peak-picking, protein database searching and reporting are all performed using an integrated
software platform and can be automated in batch mode. The instrument can be coupled with the MultiProbe and
TOFprep robotic liquid handling systems to increase sample preparation throughput. The orthogonal geometry
of the instrument allows the MALDI source to be completely decoupled from the TOF chamber, eliminating any
discrepancies associated with ionization and the sample target. This geometry enables the use of collisional ion
cooling to focus ions from the source. As a result, the ions are equilibrated so that their energy distribution is
reduced, enhancing resolution. This design eliminates the need for delayed extraction and provides enhanced
mass accuracy and stability. Mass accuracies of 10 ppm or less over an entire 384 well plate are typical using a
single external calibration. This instrument configuration also provides higher sensitivity and better resolution over a
wider mass range. Sub-femtomole BSA digests provide sequence coverage greater than 30%.