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Advantages of ReadyAgarose Precast Gels Over 96-Well Handcast Gels for High-Throughput Analysis, Rev A

the mixture in a microwave oven to dissolve the agarose. The time required to melt the agarose was noted. The liquid was allowed to cool for 10 min, and 14.5 l of 10 mg/ml ethidium bromide was added. The molten agarose was then poured into a 25 x 15 cm tray enclosed in a gel caster. Two combs, each with 51 wells, were placed in the tray. The agarose was then allowed to solidify and the time for this process was noted.

Preparing and Loading the Samples
Samples were prepared for electrophoresis by mixing 1.5 l of a 500 bp PCR sample, 3.0 l of 5x sample loading buffer containing Orange G, and 10.5 l of 1x TAE buffer for each sample. The samples were loaded into the 102-well handcast gel and two 2 x 32-well, TAE 3% ReadyAgarose gels (with ethidium bromide) using a multichannel pipet. Several lanes on each gel were loaded with Bio-Rads EZ Load 100 bp or 500 bp ruler as reference standards.

Running the Gels
The 102-well gel was run in a Sub-Cell Model 96 cell at 100 V using the PowerPac 300 power supply. The two ReadyAgarose gels were run simultaneously in two wide Mini-Sub Cell GT systems, also at 100 V. TAE buffer (1x) was used to run all of the gels, and the volume of buffer used for each run was noted. In both cases the gels were run until the Orange G dye migrated to 3.5 cm from the well. The running times were noted for both the handcast and precast gels.

Imaging the Gels
Bio-Rads Fluor-S imager and Quantity One 1-D image analysis software were used to image the gels. The handcast gel was imaged as one large gel. The ReadyAgarose gels were imaged as a composite.


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