Abstract
Genome analysis using pulsed-field gel electrophoresis (PFGE) has been used in applications ranging from typing bacterial strains to radiobiology to cancer research. While methods for running PFGE have been significantly improved since its invention, the method for preparing chromosomal DNA itself has remained essentially unchanged. This limits the applicability of PFGE, especially when analyses require many samples. We have streamlined sample preparation for routine applications of PFGE through the use of deep-well 48-well plates. Besides saving time, our protocol has the added advantage of reducing the volume of expensive reagents. Our improved protocol enables us to reduce throughput time and simplify the procedure, facilitating wider application of PFGE-based analyses in the laboratory.
Highlights
Pulsed-field gel electrophoresis (PFGE) enables large DNA molecules, including whole chromosomes, to be separated and visualized [1,2]
PFGE is routinely used in the clinical laboratory to fingerprint restriction-fragmented bacterial genomes, especially those of epidemiological interest [3,4], and to karyotype eukaryotic genomes for genetic screening [5]
After the addition of Low-melting point (LMP) agarose, resuspend the cells for 1 min using a vortex with plate adapter
Summary
Pulsed-field gel electrophoresis (PFGE) enables large DNA molecules, including whole chromosomes, to be separated and visualized [1,2]. We present a streamlined method for preparation of chromosomal DNA for pulsed-field gel electrophoreses. The main distinction between our method and standard PFGE preparation is in using one vessel (a single well on a 48 deep-well plate) to grow cultures, prepare agarose plugs, process plugs in batches, and load the gel, without having to handle tubes and DNA plugs separately or to insert individual plugs into gel wells. Streamlined preparation of genomic DNA in agarose plugs for pulsed-field gel electrophoresis.
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