BigDye® Direct Sanger Sequencing Kit | Thermo Fisher Scientific

Fewer Sequencing Steps

The BigDye® Direct Cycle Sequencing Kit, the latest innovation in the BigDye® product family, simplifies industry-standard Sanger sequencing workflow by combining post-PCR clean-up and cycle sequencing into a single step.

" Fantastic kit. The workflow is very simple and skips the post-PCR purification step. Single-stream processing protects sample integrity and identity which is very important in our environment. The kit is priced well.”
— Andrew Hadd, Senior Scientist at Asuragen, Inc.

Excellent 5' Resolution with BigDye Direct Cycle Sequencing Kit and POP-7™

Schematic overview of the SSCP technique

Benefits

Accuracy of Data: By performing all steps from PCR to capillary electrophoresis in a single tube, reduce the possibility of introducing experimental variability
Quality of Sequencing: Read as close as one base after the gene specific primer sequence with the use of POP-7™ polymer
Simplified Sequencing Workflow: Reduce the number of manual handling steps and save time. M13 universal sequencing primers (forward and reverse) are incorporated into kit, reducing need to procure multiple reagents
Time Saving: Obtain sequence reads up to 40% faster and with less hands-on time using BigDye Direct Cycle Sequencing Kit.

BigDye® Direct
Cycle Sequencing Kit

3500 Series Genetic Analyzer

Downloadable version

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
BigDye® Direct PCR Master Mix	4°C	refer to expiration date
BigDye® Direct Sequencing Master Mix	-20°C	refer to expiration date	5 cycles
BigDye® Direct Sequencing Master Mix	4°C	4 weeks
BigDye® Direct Sequencing Primers (fwd/Rev)	-20°C	refer to expiration date	5 cycles
BigDye® Direct Sequencing Primers (fwd/Rev)	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
Amplified PCR	4°C	16 hrs
Formulated Sequencing reaction mix	Ambient	16 hrs
Sequencing products (no clean up)	-20°C	4 weeks
Sequencing products (no clean up)	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

Customers will need to download a BigDye Direct Cycle Sequencing Kit Installer to update the mobility files.

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

Workflow

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