I know that Broad has an automated system for breaking emulsion PCRs and JGI is working on one as well. Bruce Roe's group has a centrifuge-based one that we've tried but it ends up being more time-consuming than the syringes.
Any others? There must be a better way than syringes...
Subscribe to:
Post Comments (Atom)
Automating The emPCR Resuspension Process For The Roche/454 GSFLX
ReplyDeleteChris L. Wright , Lorie Hetrick , Adrienne Lane , Evette Vlach , Shahjahan Ali , Jyothi Thimmapuram , Jenny Drnevich , Mark A. Mikel , Hans Bohnert
W.M. Keck Center for Comparative and Functional Genomics, Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign Urbana, IL 61801, USA
Bead recovery after emulsification and amplification is a critical step in emulsion PCR (emPCR). Resuspension of the beads after bulk emPCR amplification currently requires the manual use of several individual syringe/needle assemblies to remove the bead emulsion well-by-well out of the 96-well PCR reaction plate. Resuspension is the most intensive and rate-limiting step in the emulsion breaking process. As a result of the bench-time and labor required to aspirate the emulsion, it is the current bottleneck for high-throughput processing of samples for the GSFLX Genome Sequencer. Using the Robbins HydraTM 96, a 96-syringe liquid handling instrument, we have modified the emPCR protocol for quick and automated resuspension of the amplified emPCR beads. This facilitates emulsion transfer from PCR reaction plates into pooled emulsions in syringes in less than 20 minutes. The user will find the emulsions easier to pass through the Swinlok filter units and will have equivalent or improved recovery compared to those resuspended using the current Roche protocol. This procedure can be used on bulk plates or plates with mixed samples. Analyzed results of samples sequenced after HydraTM recovery versus manual recovery show no significant differences for average read length, percent pass filter, or other quality metrics