BigDye® Direct Cycle Sequencing Reagents-CG
Diagnostic kit development and laboratory use with confidence and compliance
The BigDye® Direct Cycle Sequencing Reagents-CG, the latest innovation in the BigDye® product family, which simplify a workflow based on gold-standard Sanger sequencing, by combining post-PCR clean-up and cycle sequencing into a single step. These products are now available under general purpose reagent (GPR) labeling for developing your diagnostic assay and for use in your laboratory.
Accuracy of data
By performing all steps from PCR to capillary electrophoresis in a single tube, you can reduce the possibility of introducing experimental variability.
Read as close as one base after the gene specific primer sequence with the use of POP-7™ polymer. Simplified sequencing workflow- M13 universal sequencing primers (forward and reverse) are incorporated into the kit, to help reduce the number of manual handling steps.
Obtain sequence reads up to 40% faster and with less hands-on time using BigDye® Direct Cyle Sequencing Reagents-CG.
You can be confident in using these materials for your applications as unlike RUO-labeled products, these products have been manufactured in accordance with FDA requirements for GPRs (General Purpose Reagents), without changing the existing formulation.
Q: Which plate format is compatible with BigDye® Direct Cycle Sequencing Reagents-CG?
A: 96-well and tubes (strip tubes or single tubes).
Q: Can customers combine other PCR mixes to use with the BigDye® Direct Cycle Sequencing Reagents-CG?
A: No, all components of BigDye® Direct Cycle Sequencing Reagents-CG are specially formulated and optimized to work together, and cannot be combined with other PCR master mixes.
Q: Can I use standard gene specific PCR primers?
A: No, you are not able to use standard gene specific PCR primers with the BigDye® Direct Cycle Sequencing Kit. You are required to use universal primers, specifically M13 universal primers. Here are the universal M13 sequences for primer design:
- M13 forward primer sequence: 5’ TGTAAAACGACGGCCAGT 3’
- M13 reverse primer sequence: 5’ CAGGAAACAGCTATGACC 3‘
Q: Can I use standard gene-specific sequencing primers?
A: No, you are not able to use standard gene-specific sequencing primers with the BigDye® Direct Cycle Sequencing Reagents-CG. You are required to use the M13 universal primers included in the package.
Q: What is the recommended template DNA concentration, pre-PCR?
A: The recommended DNA concentration is 4 ng/µL.
Q: Is it preferable to use fresh PCR product or stored product for use with BigDye® Direct Cycle Sequencing Reagents-CG? If stored, what temperature conditions should be used?
A: It is always recommended to use fresh PCR product, but if there is a need to store it, please refer to question on storage conditions.
Q: Is it better to store samples dry or in Hi-Di™ formamide? What is the best temp?
A: It is recommended to store samples dry at -20°C.
Q: Can customers dilute the BigDye® Direct Cycle Sequencing Reagents-CG?
A: The BigDye® Direct Cycle Sequencing Reagents-CG formulation has been optimized to work at full strength. Diluting the product will result in poor results.
Q: What dye set is used for the spectral calibration?
A: Please use the BigDye® Terminator v3.1 Long Read Sequencing Standard to spectrally calibrate the instrument for dye set Z.
Q: What can be done if pull-up peaks are experienced, caused by off-scale data?
A: It is recommended one of the following efforts be tried:
- Reduce the number of PCR cycles by 2-3 cycles
- Re-inject the affected samples
- Optimize injection parameters by reducing time or voltage