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1. What component and quantities are included in the BigDye® Direct Cycle Sequencing. Answer
| | Kit Component and Quantity |
| P/N | BigDye® Direct
PCR Master Mix | BigDye® Direct
Sequencing Mix | BigDye Direct
M13 Forward
Primer | BigDye® Direct
M13 Reverse
Primer | DNA Control |
| 4458689 | 24 rxn | 24 rxn | 24 rxn | 24 rxn | Sufficient for 200 rxn |
| 4458687 | 100 rxn | 100 rxn | 100 rxn | 100 rxn | Sufficient for 200 rxn |
| 4452688 | 100 rxn | 100 rxn | 100 rxn | 100 rxn | Sufficient for 200 rxn |
2. On what platform is the BigDye® Direct Cycle Sequencing Kit supported? Answer
| Instrument | OS | Data Collection |
| 3130/XL | XP SP2 | 3.0 and 3.1 |
| 3500/XL (DX as well) | Vista SP1 | 1.0 |
| 4452688 | XP SP2 | 3.0 and 3.1 |
| Secondary Analysis Software | Version |
| Seq A | 5.4, 5.3.1 and 5.3 |
| SeqScape | 2.7 and 2.6 |
| Variant Reporter | 1.1 and 1.0 |
3. With what plate format is the BigDye® Direct Cycle Sequencing Kit compatible? Answer
96-well and tubes (strip tubes or single tubes).
4. What are the BigDye® Direct Cycle Sequencing Kit storage conditions? Answer
| Description | Storage
Conditions | Stability | Freeze Thaw
-20°C← →Ambient |
BigDye® Direct
PCR Master Mix | -20°C | refer to expiration date | 5 cycles |
| 4°C | refer to expiration date | |
BigDye® Direct
Sequencing Master Mix | -20°C | refer to expiration date | 5 cycles |
| 4°C | 4 weeks | |
BigDye® Direct
Sequencing Primers (fwd/Rev) | -20°C | refer to expiration date | 5 cycles |
| 4°C | 4 weeks | |
| Control CEPH DNA | -20°C | refer to expiration date | N/A |
*if the BigDye Direct Cycle Sequencing Kit has been left at room temp for >8 hours, it is recommended that the customer does not use the kit.
5. Under what conditions is the DNA stable in each of the BigDye Direct Cycle Sequencing steps? Answer
| Description | Storage Conditions | Stability |
| Formulated PCR reaction mix | Ambient | 16 hrs |
| Amplified PCR | -20°C | 4 weeks |
| 4°C | 16 hrs |
| Formulated Sequencing reaction mix | Ambient | 16 hrs |
| Sequencing products (no clean up) | -20°C | 4 weeks |
| 4°C | 16 hrs |
| Sequencing product (after BDx clean up) | -20°C | 10 days |
| 4°C | 10 days |
| Ambient | 16 hrs |
6. Can customers combine other PCR mixes to use with the BigDye Direct Cycle Sequencing Kit? Answer
No, all components of BigDye Direct Cycle Sequencing kit are specially formulated and optimized to work together, and cannot be combined with other PCR master mixes.
7. Can customers use standard gene specific PCR primers? Answer
No, customers are not able to use standard gene specific PCR primers with the BigDye Direct Cycle Sequencing Kit. Customers are required to use universal primers, specifically M13 universal primers. Here is the sequence:
M13 universal sequences for primer design
- M13 forward primer sequence: 5’ TGTAAAACGACGGCCAGT 3’
- M13 reverse primer sequence: 5’ CAGGAAACAGCTATGACC 3'
8. Can customers use standard gene specific sequencing primers? Answer
No, customers are not able to use standard gene specific sequencing primers with the BigDye Direct Cycle Sequencing Kit. Customers are required to use the M13 universal primers included in the kit.
9. What is the recommended template DNA concentration, pre-PCR? Answer
The recommended DNA concentration is 4 ng
10. Under what temperature and time conditions is the DNA from the sample stable in the BigDye XTerminator® Purification Kit? Answer
Samples can be stored in BigDye Xterminator Purification Kit, please refer to question #4 for storage conditions.
11. Is it preferable to use fresh PCR product or stored product for use with BigDye DirectCycle Sequencing Kit?If stored, what temperature conditions should be used? Answer
It is always recommended to use fresh PCR product, but if there is a need to store it, please refer to question #4 for storage conditions.
12. Is it better to store samples dry or in Hi-Di™ Formamide? What is the best temp? Answer
It is recommended to store samples dry at -20°C.
13. Can customers dilute the BigDye Direct Cycle Sequencing Kit? Answer
The BigDye Direct Cycle Sequencing Kit formulation has been optimized to work at full strength. Diluting the product will result in poor results.
14. Is an update to current Data Collection Software or Secondary Analysis Software needed? Answer
15. What Dye Set is used for the Spectral Calibration? Answer
Please use the BigDye Terminator v3.1 Long Read Sequencing Standard to spectrally calibrate the instrument for dye set Z.
16. What can be done if pull-up peaks are experienced, caused by off-scale data? Answer
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
