PERPENDICULAR GRADIENT GEL ANALYSIS
A 7.5 x 10 cm, 1 mm thick, 25% to 65% denatured gradient gel was made using 6% aclylamide/bis (37.5:1) in 1x TAE buffer (50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA). Four different PCR products each from exon 5, 6, 7 and 8 were mixed in a 25 l volume and the total sample volume made up to 100 l. We added 100 ml 2x gel loading dye (70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 2 mM EDTA) to these samples, and electrophoresed them on the DCode system at 130 V for 2.5 hours at 56 C. After electrophoresis, the gels were stained in a 1:10,000 dilution of SYBR Green I (Molecular Probes) in 1x TAE buffer for 45 minutes and destained in 1x TAE buffer for 45 minutes. The gels were imaged under UV transillumination.
PARALLEL GRADIENT GEL ANALYSIS
A 16 x 16 cm, 1 mm thick, 45% to 60% denatured gradient gel was made using 6% aclylamide/bis (37.5:1) in 1xTAE buffer. Five ml of PCR products (approximately 200300 ng) was mixed with 5 l 2x gel loading dye and electrophoresed on the DCode system at 150 V for 3.5 hours at 60 C. The post-run analysis was the same as perpendicular gradient gel analysis.
Results and Discussion
The optimum concentration of denaturant to be used for parallel gradient gels was determined from the perpendicular gradient gel analysis. All exons separated and had a melting transiti