Loading buffer was prepared by combining deionised formamide and 25 mM EDTA (pH 8.0) with 50 mg/ml blue dextran in a 5:1 ratio. Loading buffer was stored at -25℃C.
Each sample pellet was resuspended in 3 µl of loading buffer, vortexed briefly and spun. The samples were heated at 95℃C for 2 minutes to denature and then placed on ice prior to loading. The loaded volume per sample depended on sequencing chemistry and comb sizes. A range from 0.7 µl (BigDye Terminator, 96 well gels) to 1.5 µl (Dye Primer, 36 well gels) was used.
© 2001 Alexander Binder