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CastAway Precast Gels for Rapid Automated DNA Sequence Analysis

s: assemble the gel apparatus, mix reagents, degas the gel solution, pour the gel, and complete gel polymerization. In contrast, Stratagenes CastAway gels are provided polymerized and ready to load in less than 30 minutes (Figure 1).

The gel apparatus and precast gel are easily assembled in four steps (Figure 1). First, remove the CastAway gel from its packaging and allow it to equilibrate to room temperature. Second, rinse the front and back glass plates with distilled water and dry with lint-free wipes. Third, tighten the left and right precast gel rails and the upper buffer tank into position, and the gel assembly is complete. Finally, install the gel apparatus on the LI-COR instrument and carry out a 20 minute pre-electrophoresis prior to loading the gel.

Focusing and Electrophoresis

The LI-COR IR2 Automated DNA Analyzer uses a scanning infrared fluorescence microscope to detect the electrophoretic movement of fluorescently labeled DNA molecules in real time. The system is a two-dye infrared automated DNA sequencer that can detect the products of two sequencing reactions in parallel by generating two separate gel images, each corresponding to the fluorescence emission of a different infrared dye (700 nm channel for IRD 700; 800 nm channel for IRD 800).

The microscope consists of one or two laser diodes for providing dye excitation (wavelength at 685 nm and 785 nm) and one or two avalanche photodiodes for detecting the fluorescence emission.6,7 By using infrared radiation, the background fluorescence caused by the soda lime glass of the precast gel is greatly reduced. Moreover, variations in precast gel thickness can be accommodated by autofocusing the fluorescence microscope to
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Mol wt: monoisotopic mol wt1,045.5423 Da by calculation...
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