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View additional product information for BigDye™ Terminator Plus Cycle Sequencing Kit - FAQs (4337466, 4337465, 4337463, 4337462, 4337461)
14 product FAQs found
Yes, sequencing reactions can be diluted when using the BigDye Terminator Plus Cycle Sequencing Kit by using the 5X Sequencing Buffer. When reducing the Ready Reaction Mix volume, additional buffer should be added to maintain a final 1X buffer concentration.
The 5X Sequencing Buffer contains buffer components (for example, Mg²⁺ and Tris) at a higher concentration than the Ready Reaction Mix.
Specifically, the Ready Reaction Mix provides buffer at 2.5X, while the Sequencing Buffer is at 5X, meaning the buffer components are twice as concentrated in the 5X Sequencing Buffer compared to the Ready Reaction Mix. To maintain a final 1X buffer concentration in the sequencing reaction when reducing the volume of Ready Reaction Mix, the volume of 5X Sequencing Buffer added should compensate for the missing buffer. This can be calculated as:
Volume of 5X Sequencing Buffer = ½ × (Nominal Ready Reaction Mix volume – Actual Ready Reaction Mix volume)
Example: For a 10 µL sequencing reaction:
Nominal Ready Reaction Mix volume (full reaction): 4 µL
Actual Ready Reaction Mix volume used: 1 µL
Volume of 5X Buffer = ½ × (4 µL – 1 µL)
Volume of 5X Buffer = ½ × 3 µL = 1.5 µL
In this example, adding 1.5 µL of 5X Sequencing Buffer compensates for the reduced Ready Reaction Mix volume and maintains the correct final buffer concentration for optimal sequencing performance.
This helps to ensure that the overall buffer composition remains consistent despite dilution of the sequencing chemistry.
Yes, BigDye Terminator Plus Cycle Sequencing Kit can be used with dGTP chemistry when sequencing challenging templates. When using dGTP, standard reaction setups can be followed, with the addition of a small proportion of dGTP BigDye master mix to the sequencing reaction. Optimization may be required depending on the template.
Yes, BigDye Terminator Plus Cycle Sequencing Kit is compatible with commonly used sequencing additives, including GC enhancers, DMSO, betaine, sulfolane, and sequencing enhancer solutions. These additives may be used when working with particularly challenging templates.
BigDye Terminator Plus Cycle Sequencing Kit is designed to support sequencing performance across challenging templates, including GC-rich regions, repetitive sequences, and templates with secondary structure.
BigDye Terminator Plus Cycle Sequencing Kit is designed to support longer read lengths and more consistent signal across challenging templates compared to BigDye Terminator v3.1 Cycle Sequencing Kit, particularly in GC-rich or structured regions.
Yes, BigDye Terminator Plus Cycle Sequencing Kit is compatible with standard M13 primers and other sequencing primers without requiring changes to the workflow.
The BDTv3direct.mob mobility file is recommended when using BigDye M13 HiRes primers, as it is optimized for improved peak spacing and base alignment at the start of the sequence.
BigDye M13 HiRes primers have the same sequence as standard M13 primers but include a modification that supports improved resolution at the 5′ end, enhancing basecalling quality within the first few bases downstream of the priming site. Standard M13 primers remain suitable for most applications.
No, standard sequencing purification methods can be used. BigDye Terminator Plus Cycle Sequencing Kit is compatible with commonly used cleanup approaches such as magnetic beads, gel filtration, and ethanol precipitation.
Thermo Fisher Scientific has verified the following purification methods for use with BigDye Terminator Plus Cycle Sequencing Kit:
BigDye XTerminator Purification Kit
Dynabeads Sequencing Clean-Up Kit
MicroSEQ ID Ultra Sequencing Clean-up Plates Kit
Ethanol/EDTA precipitation
Other equivalent purification methods may be used as long as they effectively remove unincorporated dye terminators and salts.
A reduced-volume setup of 1 µL Ready Reaction Mix in a 10 µL reaction is the recommended starting point. For challenging samples, increasing the Ready Reaction Mix volume may help improve signal intensity (for example, 4 µL in a 10 µL reaction).
The BigDye Terminator Ready Reaction Mix contains the components required for fluorescence-based cycle sequencing, including dNTPs, fluorescently labeled ddNTPs, buffer components, and the sequencing enzyme. The premixed formulation is supplied as a concentrated reagent to allow flexibility in reaction setup; the exact formulation is proprietary.
BigDye Terminator Plus Cycle Sequencing Kit is designed to support high-quality sequencing results with minimal changes to standard Sanger sequencing workflows. It supports performance on challenging templates, including GC-rich regions, repetitive sequences, and flexible reaction volumes, helping maintain consistent signal and read continuity, and it has compatibility with fast cycling protocols to increase efficiency and optimize the workflow without compromising data quality.
BigDye Terminator v1.1 Cycle Sequencing Kit is commonly used for improved resolution near the primer (5′ region), particularly when used with POP-6 polymer. BigDye Terminator v3.1 Cycle Sequencing Kit is suitable for general sequencing of larger templates.
BigDye Terminator Plus Cycle Sequencing Kit is designed to support improved performance on challenging templates, such as GC-rich or structured regions, while maintaining long read lengths. It helps improve 5′ region and basecalling within the first few bases downstream of the M13 priming site when used with POP-7 polymer and BigDye Hi-Res M13 primers.