DOE Human Genome Program Contractor-Grantee
41. Automation Using Packard Multiprobe Robots for Finishing
Christine Munk, Judy Buckingham, Marie Krawczyk, Elizabeth Saunders, David Bruce, and Mark Mundt
Bioscience Division and DOE Joint Genome Institute, Los Alamos National Laboratory, Los Alamos, NM 87545
We have recently adopted an automated approach for cherry-picking of subclone DNA to streamline our finishing process. The input for this process is a list of subclones for finishing reactions that is generated using the Phrap .ace file. A script sorts the resequencing reactions by single strand gap size and calculates source and destination for each subclone DNA. The script outputs two text files, one of which is formatted to be imported into an MP Table sample transfer program and contains source and destination locations for the DNA transfer. The second output file is used to make the sample sheet, which is imported into the data collection software on the ABI 377.
For cherry-picking subclones, we initially tried a Packard Multiprobe I equipped with disposable tips to transfer DNA from deepwell cluster tubes. We missed ~20% of the DNAs because the edge near the top of the tips got hung up on the top of the DNA tube, preventing the tip from reaching the DNA at the bottom of the tube. To address this problem, we moved to a fixed-tip robot, and the DNA prep procedure was changed to collect DNA into microtiter plates. The small diameter of the fixed tips allows them to reach the bottom of both the deepwell and microtiter plates. In our current process, we use a fixed-tip Multiprobe I robot with MP Table software to transfer subclone DNAs from round-bottom microtiter plates and to rearray these into cycleplates for sequence reaction setup. We have not experienced problems with DNA cross-contamination using a simple water rinse of the tips between samples. The cost of disposable tips has been eliminated. Use of microtiter plates instead of deepwells has greatly reduced the amount of storage space needed.
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