Efficacy Of Using A Combination Microplate Washer For Vacuum-Based DNA Sequencing Reaction Cleanup

The ability to determine the specific pattern of base pairs in DNA molecules is an indispensable part of contemporary molecular biology. Over the past 10-12 years the evolution of market leading dye terminator methods and automated capillary electrophoresis instrumentation has largely standardized the procedure for DNA sequencing, quickly making it more accessible, less resource intensive, and easier to perform at many different throughput levels. A critical component of this genomic workflow is the sequencing cleanup procedure, where contaminating artifacts of the sequencing reaction are removed prior to capillary electrophoresis. There are currently a number of viable DNA sequencing cleanup methods available using either filtration, precipitation, or sequestering. Each method has its own costs and benefits and is a proven way of purifying reaction samples. In collaboration with a comprehensive DNA Analysis Core Facility that utilizes state-of-the-art sequencing chemistires and technology, a microplate washer fitted with an integrated vacuum filtration module was used to perform DNA sequencing reaction cleanup. Results were substantiated against a gel filtration method currently used by the collaborator. Evidence provided by this demonstration support the efficacy of the microplate washer demonstrated here to contribute to the genomic workflow typical of many molecular biology laboratories and core facilities.

Two separate runs were completed. Run #1 was designed to gauge BioTek vacuum performance using the Millipore kit recommended protocol, and compare any differences between manual pipetting and shaking for the resuspension step. Run #2 optimized results observed from the first run. Post cycle sequencing pGEM samples were divided between the gel filtration plate and the vacuum filtration plate. For quality control purposes the injection plate protocol was defined to process one set of gel filtration samples first, followed by the vacuum samples, and finish with a final group of gel filtration samples. Figure 3 shows the complete workflow for the experiment.