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BigDye™ Direct Cycle Sequencing Kit, 100 reactions - FAQs

BigDye™ Direct Cycle Sequencing Kit, 100 reactions - FAQs

View additional product information for BigDye™ Direct Cycle Sequencing Kit - FAQs (4458689, 4458688, 4458687)

29 product FAQs found

What do I do if I have pull-up peaks caused by off-scale data when using the BigDye Direct Cycle Sequencing Kit?

We recommended that you perform one of the following:

- Reduce the number of PCR cycles by 2–3 cycles
- Re-inject the affected samples
- Optimize injection parameters by reducing time or voltage

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Do you have any recommendations for the pre-PCR template DNA concentration in the BigDye Direct Cycle Sequencing Kit procedure?

The recommended DNA input amount is 4 ng.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Am I required to use fresh PCR product with the BigDye Direct Cycle Sequencing Kit, or can I store the PCR reaction before proceeding? What temperature is best for storage?

We always recommended that you use fresh PCR product, but if you need to store it, the amplified PCR reaction can be stored for 16 hours at 4°C or up to 4 weeks at –20°C.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Can I store samples in BigDye Xterminator solution? If so, what is the optimal temperature, and for how long can I store it?

Yes, after cleaning the sequencing product using the BigDye XTerminator Purification Kit, the end product can be stored up to 10 days at either 4°C or –20°C.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

If I need to pause my experiment in the middle of the BigDye Direct Cycle Sequencing Kit procedure, how long can I expect my samples to be stable?

The amplified PCR reaction can be stored for 16 hours at 4°C or up to 4 weeks at –20°C. The formulated sequencing reaction mix can be stored at ambient temperature for up to 16 hours. After cycle sequencing but before cleanup, the sequencing product can either be stored for up to 16 hours at 4°C or up to 4 weeks at –20°C. After cleaning the sequencing product up using the BigDye XTerminator Purification Kit, the end product can be stored up to 10 days at either 4°C or –20°C.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What are the BigDye Direct Cycle Sequencing Kit storage conditions?



BigDye Direct PCR Master Mix: can be stored at 4°C or –20°C. Refer to the expiration date on label for expiration details. The BigDye Direct PCR Master Mix can undergo up to 5 freeze-thaw cycles (–20°C to ambient)

BigDye Direct Sequencing Master Mix: can be stored at 4°C for up to 4 weeks or at -20°C up to the expiration date on the label. The BigDye Direct Sequencing Master Mix can undergo up to 5 freeze-thaw cycles (–20°C to ambient)

BigDye Direct Sequencing Primers (fwd/rev): can be stored at 4°C for up to 4 weeks or at –20°C up to the expiration date on the label. The BigDye Direct Sequencing Primers can undergo up to 5 freeze-thaw cycles (–20°C to ambient)

Control CEPH DNA: can be stored at –20°C. Refer to the expiration date on the label for expiration details.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What software platforms are the BigDye Cycle Sequencing Kits supported on?

The analysis modules are compatible with Sequencing Analysis Software v5.3, 5.3.1, and 5.4, SeqScape Software v2.6 and v2.7, and Variant Reporter v1.0 and v1.1. If the Analysis Software is running on an instrument computer, the instrument also needs to be running either Data Collection v3.0 or 3.1 (Applied Biosystems 3130 or 3730 DNA Analyzers) or Data Collection v1.0 (Applied Biosystems 3500 DNA Analyzers).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Which high-throughput plate formats are the BigDye Direct Cycle Sequencing Kits compatible with?

The BigDye Direct Cycle Sequencing Kits are compatible with 96-well plates and tubes (strip tubes or single tubes).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Can I dilute the reagents in the BigDye Direct Cycle Sequencing Kit?

The BigDye Direct Cycle Sequencing Kit formulation has been optimized to work at full strength. Diluting the product may lead to poor results.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Do I need to update my current instrument Data Collection Software or Secondary Analysis Software for use with BigDye Direct Cycle Sequencing kits?

It depends on the version of software. The analysis modules are compatible with Sequencing Analysis Software v5.3, 5.3.1, and 5.4, SeqScape Software v2.6 and v2.7, and Variant Reporter Software v1.0 and v1.1. It will be necessary to download the BigDye Direct Installer for these versions. If the Analysis Software is running on an instrument computer, the instrument also needs to be running either Data Collection v3.0 or 3.1 (Applied Biosystems 3130 or 3730 DNA Analyzers) or Data Collection v1.0 (Applied Biosystems 3500 DNA Analyzers). If Sequencing Analysis Software v6, SeqScape Software v3, and Variant Reporter Software v2 are being used, the BigDye Direct settings are present.

To update your mobility files, go to our software download page by navigating to www.thermofisher.com/technicalresources, click on “Software, Patches & Updates”, and under DNA Sequencing look for “BigDye Direct Cycle Sequencing Kit”.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Can I combine other PCR mixes to use with the BigDye Direct Cycle Sequencing Kit?

No, all components of the BigDye Direct Cycle Sequencing kit are specially formulated and optimized to work together and they cannot be combined with other PCR master mixes.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What is a dye blob?

After the cycle sequencing reaction is complete, unincorporated ddNTPs can form complexes that migrate more slowly than individual ddNTPs would. These complexes can interfere with electrokinetic injection, electrophoretic separation, and data analysis. In the electropherogram these can be observed as a region containing multicolored peaks of varying sizes or blobs, depending on the efficiency of the cleanup. In BigDyeTerminator v3.1 reactions, these blobs may appear around 70–80 bp and, in severe cases, 110–120 bp. For BigDye Terminator v1.1, the blobs will either migrate ahead of the data collection time (so they will not be visible) or they may be visible at the very beginning of the run.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What are the different ways to clean up my sequencing reactions?

While there are many kits and methods available to clean up the extension products from a sequencing reaction, the ones that Thermo Fisher Scientific has formally tested and recommends are:

•BigDye XTerminator Purification Kit
•Ethanol precipitation
•Spin column/plate purification (e.g. Centri-Sep columns, DTR Kits from Edge Biosystems)

A list of alternative methods for purification is available in the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C, page 119). The guide can be found by searching the Thermo Fisher Scientific website with the Cat. No. 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What is the difference between the regular BigDye (Terminator) kits and the BigDye Direct Cycle Sequencing Kit?

BigDye Direct Cycle Sequencing Kit supplies reagents for the complete cycle sequencing workflow, including the PCR reaction, reaction cleanup, and sequencing the PCR product. The post-PCR and cycle sequencing process is combined into a single step, and the workflow requires that the initial PCR primers be designed with an M13 tail, since M13 primers will be used for the sequencing reaction. BigDye Terminator Cycle Sequencing Kits are designed so that the initial PCR and the cleanup are performed as separate reactions. This allows the user more flexibility in primer design and the option to break up the workflow. For the comparative studies that we performed in-house, we found sequencing data obtained using these two kits to be concordant and of at least equivalent quality (typically the BigDye Direct Cycle Sequencing Kit produced better results).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Which kit is best for my application?

Selecting the best kit for your application depends on what you are trying to do—for example, the goals and needs for a resequencing project may be different than for SNP detection. To help guide you, Thermo Fisher Scientific has put together an Applications/Chemistry table found in the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C). The guide can be found by searching the Thermo Fisher Scientific website with the Cat.No 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Should I use the BigDye Terminator v3.1 Cycle Sequencing Kit or BigDye Direct kit if I want to directly perform cycle sequencing on long-range amplicons (up to 10kb) using PCR primers for sequencing, instead of M13 primers?

We recommend the BigDye Terminator v3.1 Cycle Sequencing Kit if you would like to use custom PCR primers instead of M13 primers for sequencing.

The BigDye Direct kit must use the M13 primers provided in the kit for sequencing, as the kit contains the PCR and Sequencing components and are optimized to work together.

How do I order custom primers with an M13 tail to use with the BigDye Direct Cycle Sequencing Kit (Cat. No. 4458687)?

You can order custom primers with an M13 tail to use with the BigDye Direct Cycle Sequencing (Cat. No. 4458687) using our online Custom DNA Oligos Synthesis Services .

If you need assistance designing your primers, you may use the OligoPerfect Primer Designer tool.

Once you have the primer sequences, you can manually add the M13 sequences described on page 10 in the BigDye Direct Cycle Sequencing Kit Protocol to the 5' ends of the PCR primer sequences and then order through Custom DNA Oligos Synthesis Services.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

Can I set up a sequencing reaction control for the BigDye Direct reaction?

The control that would be used for the sequencing reaction is the same control for the PCR reaction. For instance, in the kit, we provide the Control DNA CEPH 1347-02; this control should be taken into the PCR reaction and sequenced. Please see here (https://tools.thermofisher.com/content/sfs/manuals/cms_091494.pdf) for setting up the PCR reaction and the sequencing reaction.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Do I need different mobility and basecaller files for the BigDye Direct samples or can the BigDye Terminator files be used?

The BigDye Direct samples have specific mobility and basecaller files depending on the instrument.

-KB_3130_POP7_BDTv3direct.mob and KB_3130_POP7_BDTv3direct.bcc
-KB_3500_POP7_BDTv3direct.mob and KB_3500_POP7_BDTv3direct.bcc
-KB_3730_POP7_BDTv3direct.mob and KB_3730_POP7_BDTv3direct.bcc


Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What is the PCR cleanup method used in the BigDye Direct Cycle Sequencing Kit?

It is an enzymatic cleanup and, due to the proprietary nature of the kit, we cannot disclose additional information.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What is the sequence of the M13 tails to be added to my PCR primer when using the BigDye Direct Cycle Sequencing Kit?

-M13 forward primer sequence: 5′ TGTAAAACGACGGCCAGT 3′
-M13 reverse primer sequence: 5′ CAGGAAACAGCTATGACC 3′

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Why do my target-specific PCR primers need the M13 forward and M13 reverse tails when using the BigDye Direct Cycle Sequencing Kit?

The M13 tails are necessary as the BigDye Direct Cycle Sequencing Kit contains the universal M13 forward and universal M13 reverse primer, without the M13 tails the sequencing reaction will fail. The use of M13 sequencing primers also reduces the loss of valuable 5' unresolvable bases.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Can I dilute the BigDye Direct Cycle Sequencing Kit?

No, the BigDye Direct Cycle Sequencing Kit formulation has been optimized to work at full strength and the PCR and sequencing chemistry is optimized to work together for a simpler workflow. Diluting the product may lead to poor results.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

In the PCR amplification of BigDye Direct Cycle Sequencing Kit, it uses an annealing temperature of 62 degrees C. Does this apply to all primers?

The PCR guidelines in the BigDye Direct Cycle Sequencing Kit manual are general guidelines designed to work for a wide variety of samples. However, there will be cases where the PCR conditions may need to be modified (e.g. Tm of primer is very high, GC rich products, etc.) to best work with whatever you are trying to amplify and sequence. The primers must have the M13 tail on the 5' end. For further suggestions, see Page 38 in the DNA Sequencing by Capillary Electrophoresis Guide (http://tools.thermofisher.com/content/sfs/manuals/cms_041003.pdf). Some of those suggestions include:

- Primers should be at least 18 bases long (not including the M13 tail which is not part of the PCR reaction)
- Tm of the primers should be 45 degrees C or higher
- Avoid runs of identical nucleotides, especially runs of 4 or more "Gs"


Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Can I use standard gene-specific sequencing primers with the BigDye Direct Cycle Sequencing Kit?

No, the M13 universal primers must be used, and are included in the BigDye Direct Cycle Sequencing Kit.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Can I use standard gene-specific PCR primers with the BigDye Direct Cycle Sequencing Kit?

No. The gene-specific PCR primers used in the BigDye Direct Cycle Sequencing Kit must be modified with the M13 universal primers on the 5' end.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Can I use the BigDye Direct Cycle Sequencing Kit on any organism?

The BigDye Direct Cycle Sequencing Kit can be used on any organism. However, we only supply Human CEPH-1347-02 control DNA in the kit. A control for your organism should be obtained from an appropriate vendor.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Can I use the BigDye Direct Cycle Sequencing Kit with my own PCR master mix?

Components of the BigDye Direct Cycle Sequencing Kit are specially formulated and optimized to work together and they cannot be combined with other PCR master mixes. Other PCR master mixes are not recommended as they can introduce variability into the workflow and yield suboptimal results.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What is the BigDye Direct Cycle Sequencing Kit?

The BigDye Direct Cycle Sequencing Kit streamlines the workflow when sequencing PCR products. The kit combines the components for the PCR reaction, PCR purification and cycle sequencing, which results in substantial time savings.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.