BrightDye™ Terminator Cycle Sequencing kit - Quick Start Guide The BrightDye™ Terminator Cycle Sequencing kit is based on the Sanger Sequencing method, delivering fluorescent labeled fragments, to be separated and analyzed using a suitable Electrophoresis platform (ABI 310, 31xx, 3500 and 3730 series Genetic Analyzers). All reagents are provided in a ready-reaction, premixed format: Just add template DNA and Sequencing primer. The kit includes 5x BrightDye™ Sequencing buffer, optimized for BrightDye™ mixes. Template Quantity PCR product: 100 - 200 bp PCR product:200 - 500 bp PCR product: 500 - 1000 bp PCR product: 1000 - 2000 bp PCR product: >2000 bp Single-stranded Double-stranded Cosmid, BAC Bacterial Genomic DNA 1-3 ng 3-10 ng 5-20 ng 10-40 ng 20-50 ng 25-50 ng 150-300 ng 0.5-1.0 µg 2-3 µg General Reaction (1x rxn) BrightDye™ Reaction Premix Template DNA Sequencing Primer Water 8 µl See table 3-5 pmole Add up to 20 µl General Reaction Setup in 10 µl End Volume (0.5 rxn) BrightDye™ Reaction Premix Template DNA Sequencing Primer Water 4 µl See table 3-5 pmole Add up to 10 µl Use of BrightDye™ 5x Sequencing Buffer The 5x concentrated BrightDye™ Sequencing buffer can be used to lower the amount of the BrightDye™ Reaction Premix. Since the concentration of the Sequencing Buffer (5x) is twice the concentration to the BrightDye™ Reaction Premix (2.5x), just use 0.5 µl of 5x Buffer to compensate for 1 µl of BrightDye mix Example for a 1/8 rxn (10 µl End Volume, where 3 µl of BrightDye React. Premix is replaced by 1.5 µl 5x Seq. Buffer): BrightDye™ Reaction Premix BrightDye™ 5x Sequencing Buffer Template DNA Sequencing Primer Water 1 µl 1.5 µl See table 3-5 pmole Add up to 10 µl Purification of Sequencing products Choose an appropriate Sequencing Reaction purification method, such as Sephadex based spin colums or plates, Magnetic Particle based kits (like Nimagen’s D-Pure™ kit), according to the Manufacturer’s protocol, or perform a standard EthOH precipitation. Electrophoresis and Analysis Run the purified extension Products, using filterset Z, on a Genetic Analyzer. Analyze with KB basecaller and a BDTv3 mobility (*.mob) settings