Divya Sharma1 and Carolyn Seggerson2, 1Tech/Aid, 7700 Edgewater Drive, Oakland, CA 94621 USA, and 2Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547 USA
Running PCR samples on agarose gels is a routine yet timeconsuming procedure. It is common to hand cast a horizontal 96-sample gel for this purpose. A gel of such large size is difficult to cast, handle, and image, and requires a long run time. These issues can be resolved by using two of Bio-Rads 2 x 32-well ReadyAgarose precast gels instead. ReadyAgarose gels are cast in their own UV-transparent running tray with fluorescent well numbers and a ruler for easy reference. Their gel trays have autolock tabs that snap into the dedicated wide mini ReadySub-Cell GT cell, so you can simply drop one of these precast gels into place and load your samples. ReadyAgarose gels fit most non-Bio-Rad wide mini subcells as well and thus do not require equipment from a specific manufacturer. ReadyAgarose gels are available in 0.8%, 1%, and 3% agarose in TBE or TAE buffer, with or without ethidium bromide, to fit your research requirements. In addition, two 2 x 32-well ReadyAgarose gels offer more wells than a 96-sample handcast gel, run in 3045 min, and have 4.0 cm of resolving distance. These precast gels are designed to give the same results as traditional handcast gels in a fraction of the time.
Hand Casting a 96-Sample Gel
A gel was made by adding 8.7 g of Bio-Rad Certified low range ultra agarose (the same agarose used to manufacture 3% ReadyAgarose gels) to 290 ml of 1x TAE buffer and heating 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.
To run the handcast gel, 2 L of 1x TAE was required, while only 1.2 L of the same buffer was needed to run the two ReadyAgarose gels
It took 45 min to run the ReadyAgarose gels at 100 V, while it took 90 min to run the handcast gel at the same voltage
When the gels were imaged, the background of the handcast gel was not uniform, suggesting that the ethidium bromide was not distributed uniformly, probably because of the high viscosity of a 3% agarose solution (Figure 1). However, no such unevenness was seen in the ReadyAgarose gels (Figure 2)
With the numbered wells on the ReadyAgarose gel tray, documentation of lane samples was both easier and less prone to error. The ruler on the ReadyAgarose tray also facilitated easy and accurate measurement of band migration distances
Time and Cost Differences
The total time needed to prepare and run the handcast and precast gels is compared in Figure 3. A sample cost analysis for 3% gels is presented in Table 1.
In our tests, both handcast and precast gels gave equivalent band sharpness for PCR samples. However, the use of two 2 x 32-well ReadyAgarose precast gels instead of a large 96-sample (102-well) handcast gel saves preparation and running time. Two ReadyAgarose 2 x 32-well gels offer more lanes than a handcast gel designed specifically for 96 samples, thus allowing extra samples or standards to be included in a single run. Data from ReadyAgarose gels are easier to document since well numbers and a ruler are printed on the tray. These advantages of using two ReadyAgarose gels over one large handcast gel make them suitable and desirable for high-throughput laboratories where gel-to-gel consistency is important and relatively small differences in time can accumulate over the long run. Using ReadyAgarose precast gels may also be cost-effective, as this particular comparison demonstrates.
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