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DYEnamic™ ET Dye Terminator Kit (MegaBACE™)- Thermo Sequenase™ II DNA Polymerase and DYEnamic™ ET Dye Terminator Sequencing Reagents optimized for MegaBACE™ DNA Analysis System.
- Higher processivity of Thermo Sequenase™ II DNA Polymerase reduces thermal cycle times by two-fold.
- Improved salt tolerance increases robustness and enables the sequencing of less pure templates.
- Efficient incorporation of DYEnamic™ ET dye terminators and dITP, which enables long read lengths with uniform peak heights for highly sensitive and accurate base calling, even with GC-rich templates.
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DYEnamic™ ET Dye Terminator Kit (MegaBACE™)
Technical Information
DYEnamic™ ET Dye Terminator Kit (MegaBACE™) features Thermo Sequenase™ II DNA Polymerase and DYEnamic™ ET dye terminators. Thermo Sequenase™ II DNA Polymerase readily incorporates DYEnamic™ ET dye terminators and dITP for improved performance on GC-rich templates. Thermo Sequenase™ II DNA Polymerase also exhibits high processivity and tolerance to high-salt conditions.
MegaBACE™ 1500 sequencing results from a relatively impure plasmid template. A plasmid template was isolated using an alkaline lysis/microwave protocol and used in sequencing reactions without further purification. Sequencing reactions were performed using DYEnamic™ ET Dye Terminator Kit. The reactions contained 100 ng of template DNA and 5 pmol of -40 M13 forward primer. The reaction was amplified for 50 cycles. Sequencing reaction products were concentrated and purified using ethanol precipitation. The sample was electrokinetically injected into the capillary at 3 kV for 45 sec and separated via electrophoresis at 9 kV for 120 min. Although beginning with an impure template, MegaBACE™ 1500 delivered accurate base calling to greater than 650 bp.
MegaBACE™ 1500 sequencing results from a plasmid template amplified from bacterial culture using a direct PCR protocol. The amplification reaction was diluted before use and was not purified using an exonuclease/alkaline phosphatase procedure. Sequencing reactions were performed using DYEnamic™ ET Dye Terminator Kit. The reactions contained approximately 50 ng of amplicon and 5 pmol of -40 M13 forward primer. The sequencing reaction was amplified for 50 cycles, and reaction products were concentrated and purified using ethanol precipitation. The sample was electrokinetically injected into the capillary at 3 kV for 45 s and separated via electrophoresis at 9 kV for 120 min. Direct amplification is perfectly suited for capillary electrophoresis because it generates sequencing templates with normalized DNA concentrations that are free of larger DNA species. This method increases overall sample-to-sample success rates and contributes to accurate base calling.
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