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Use of the DCode System to Detect the Food-Borne Bacterial Pathogen Listeria monocytogenes

es, Hercules, CA) was added to the agarose solution at a concentration of 0.5 mg/ml before the gel was poured in the mold. The gel was run for 1 hr at 50 V, and the PCR product was visualized under UV-light. The amplified products were stored at -20 C until they were used for DGGE.

DGGE
DGGE was performed with a DCode denaturing gel electrophoresis system (Bio-Rad Laboratories, Hercules, CA). A parallel gel was prepared with a denaturing gradient ranging from 15 to 40% as described in Appendix 1 (where 100% denaturant contained 7 M urea (Bio-Rad Laboratories, Hercules, CA) and 40% formamide (Fisher Scientific, Pittsburgh, PA). The gel also contained a gradient of polyacrylamide ranging from 10 to 20%, made from a 40% stock solution of acrylamide/bisacrylamide (37.5:1) (Bio-Rad Laboratories, Hercules, CA). The gel was cast using a Bio-Rad Model 385 Gradient Delivery System (Bio-Rad Laboratories, Hercules, CA) and a non-denaturing, 6% acrylamide loading gel (Appendix 1) was cast on top of the gradient gel. The gel was placed in 0.5x strength Tris/Acetic Acid/EDTA buffer (TAE) (Bio-Rad Laboratories, Hercules, CA) equilibrated to 60 C. Five l PCR product was mixed with 5 l loading buffer and applied to the gel wells. Electrophoresis was carried out at 60 C for 15 h at 200 V. The gel was subsequently soaked for 30 min in 1/10,0000x strength SYBR Green I nucleic acid gel stain (Molecular Probes, Inc., Eugene, OR) and visualized under UV-light.


Results and Discussion
Primer pair 1055F and1406R with GC-clamp yielded a single band on agarose gel corresponding to a 400 bp DNA fragment when the PCR reaction was carried out with Pfu polymerase on DNA extracted from L. m
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