Kits & Reagents for CE Sequencing & Fragment Analysis
Find which sequencing and reaction clean-up kits are right for you.
Life Technologies continues to develop superior Sanger sequencing kits that provide a solution for all your applications and templates. The table below provides guidelines to help you choose between the three main sequencing kits.
|Sequencing Specificity||BigDye® |
|All types of sequencing||xx||xx||xx|
|High resolution close to the sequencing primer||xx||–||xx (if using POP-6™)|
|M13 tailed PCR primers and M13 sequencing primer*||xx ‡||x||x|
|Universal tail primer other than M13 primer||–||x||x|
|Long read (>650 bp)||x||xx||x|
|Mixed base (heterozygote) with ratio 10/50 to 50/50||xx||x||xx|
|GC-rich, GT-rich, difficult template †||x||x||x|
† BigDye® Direct, BigDye® Terminator V3.1, and BigDye® Terminator V1.1 will provide good sequence quality for the majority of these templates. In some cases, the dGTP BigDye® terminators have been specially designed for these difficult templates and will provide better results
‡ BigDye® Direct requires the use of M13 tailed primer for the PCR reaction. This kit contains BigDye® Direct PCR Master Mix, BigDye® Direct Sequencing Master Mix to perform the PCR cleanup and sequencing reaction in a one-step reaction, BigDye® Direct M13 Fwd Primer (5′ TGTAAAACGACGGCCAGT 3′), BigDye® Direct M13 Rev Primer (5′ CAGGAAACAGCTATGACC 3′), and Control DNA CEPH 1347-02.
* Why use M13 tailed PCR primer or other universal tail primer?
Sequencing Applications and Specific Requirements
de novo Sequencing
Sequencing kit criteria for de novo sequencing: long read.
Sequencing of PCR Product (resequencing).
Sequencing kit criteria for PCR product sequencing: read first base after the primer, mixed base detection. Using a M13 primer can be a very good solution to streamline the workflow.
NGS Sequence Confirmation
Sequencing kit criteria for methylation sequencing: mixed-base detection.
Why use M13 tailed PCR primer or other universal tailed primer?
For most large projects, it has become customary to include a standard (universal) primer tail on the PCR primers to simplify sequencing setup (Figure 1). The most common tail is the M13 sequence because it was initially used for sequencing clones constructed in the single-stranded bacteriophage M13. Potential disadvantages of using tailed PCR primers are the greater challenge in designing primers with a tail and the need for higher-quality oligonucleotides due to the increase in primer length (PCR primers).Life Technologies provides great primer design tools and an attractive price for primers up to 45 bases long.
The main advantage of using an M13 tailed primer or universal tailed primer is the simplicity of the sequencing reaction setup (Figure 2).
Primer Design Tools
|Application||Primer Design Software|
|PCR and Sequencing |
PCR and Sequencing Primers
Order PCR and sequencing primers
Which Sequencing Reaction Cleanup Product Is Right for You?
|Cleanup Method||Description & Application||Product|
|Reagent-Based Sequencing Template Cleanup||Scavenges all unincorporated BigDye® terminators. Stabilizes samples before analysis. Best for long and short fragment recovery.|
|Gel Filtration Spin Columns||Recovers DNA fragments larger than 16 base pairs. Removes > 98% of salts, NTP's and other unwanted low-molecular-weight impurities.|
Fragment analysis techniques allow the development of multiple applications on the Genetic Analyzer. For an overview of the different applications visit the Fragment Analysis page.
Life Technologies offers several kits for specific fragment analysis–based applications.
Fragment Analysis Kits
|Kits and Reagents||Applications||Associated Reagents|
|SNaPshot® Multiplex System||Genotyping, BAC fingerprint, Methylation analysis||SNaPshot® primer focus kits, GeneScan™ 120 LIZ® Size Standard|
|TrueScience™ RespiFinder® Pathogen and Viral Identification Panels|| |
Detect and differentiate up to 14 RNA viruses, 1 DNA virus, and 4 bacterial strains in a single tube responsible for acute respiratory tract infections
|GeneScan™ 600 LIZ® Size Standard v2.0|
|Applied Biosystems® KRAS and BRAF mutation analysis reagents ||Detects 12 mutations in the KRAS gene and 3 mutations in the BRAF gene. Able to detect 1-5% mutation contribution in a background of wild type genomic DNA from analytical samples.|
|AFLP® Analysis System||Genome fingerprinting for microorganism or |
Genome fingerprinting for Plant
|Custom labeled primer||Microsatellite, LOH, chimerism, ISSR analysis, SSCP, RFLP, TRFLP, MSMSA|
|When DNA fragments are labeled with:||Choose a size standards labeled with:||Recommended Size Standard||Possible Applications||Other Kits and Products|
|dR110, dR6G, dTAMRA™, dROX (Dye Set DS-02 - Filter E5)||LIZ®|| |
|5-FAM™, HEX™, NED™ (Dye Set DS-30 - Filter D)||ROX™||GeneScan™ 500 ROX™ Size Standard||Custom Fragment Analysis||Custom Labeled Primer|
|6-FAM™, VIC®, NED™ (Dye Set DS-31 - Filter D**)||ROX™||GeneScan™ 500 ROX™ Size Standard|| |
Custom Fragment Analysis
|Custom Labeled Primer|
|5-FAM™, JOE, NED™ (Dye Set DS-32 - Filter F)||ROX™||GeneScan™ 500 ROX™ Size Standard|| |
|6-FAM™, VIC®, NED™, PET® (Dye Set DS-33 - Filter G5)||LIZ®||GeneScan™ 600 LIZ Size Standard v2.0|| |