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Primer Walking
In our Primer Walking service, you provide us with DNA samples, and we use the primer walking method to analyze long nucleotide sequences.
Service Contents
Using your DNA samples, we perform multiple sequencing analyses. By designing and synthesizing primers based on the sequencing result and repeating the sequencing reactions, we proceed with the long-chain sequence analysis.
- Work Process
- Sample QC
Primer design and synthesis
Sequencing reaction
Sequencer run
Data verification
Assembled data generation - Reagents
- BigDye Terminator v3.1
- Sequencer
- 3730xl DNA Analyzer
Sequencing Methods
You can choose either single-strand sequencing (SSS) or double-strand sequencing (DSS) according to your needs.
Averaged sequencing length per one reaction is approximately 500 bases. We will repeat the several sequencing reactions according to the required sequencing length.
Example: For approximately 3.0kb of Primer Walking (SSS), we usually perform around 6 sequencing reactions (3,000bp ÷ 500bp/reaction = 6 reactions).
Turnaround Times
- * The turnaround times are estimates and may change depending on the number of samples and analysis conditions.
- * If the analysis target is a known sequence, the turnaround time can be shortened. Please consult us.
Delivery
The analysis results will be provided via web download and on a data CD-R.
| Deliverables | Delivered Data |
| – Synthesized primers – Data CD-R |
– Sequence Data (.txt) – Raw Data (.ab1) – Assembly data (.txt) – Primer information |
- * We can also report the sequence data after each sequencing reaction if desired. Please let us know if you want this service.
- * Data is deleted from the server 3 months after delivery.
Price
The total analysis price includes all necessary primer design and synthesis, sequencing, and assembly.
| Base Length | Single Strand (SSS) | Double Strand (DSS) |
| Up to1.0kbp | 22,000 JPY | 44,000 JPY |
| Up to1.5kbp | 30,500 JPY | 61,000 JPY |
| Up to2.0kbp | 39,000 JPY | 78,000 JPY |
| Up to2.5kbp | 56,000 JPY | 112,000 JPY |
| Up to3.0kbp | 64,500 JPY | 129,000 JPY |
| Up to3.5kbp | 73,000 JPY | 146,000 JPY |
| Up to4.0kbp | 81,500 JPY | 163,000 JPY |
| Up to5.0kbp | 107,000 JPY | 214,000 JPY |
| Up to6.0kbp | 124,000 JPY | 248,000 JPY |
| Up to7.0kbp | 149,500 JPY | 299,000 JPY |
| Up to8.0kbp | 166,500 JPY | 333,000 JPY |
| Up to9.0kbp | 192,000 JPY | 384,000 JPY |
| Up to10.0kbp | 234,500 JPY | 469,000 JPY |
* Prices are exclusive of tax.
Required for Analysis
|
DNA Template |
Plasmid：Conc. 100 – 300ng/μL PCR Product：Conc. 20+ ng/μL |
Refer to the below for required quantities. |
|
Sequencing Primer |
Conc. 10μM （10+ μL） Universal primers are available on request. |
|
|
Order Form |
Sequencing Service Order Form Select “Primer Walking (SSS)” or “Primer Walking (DSS)” in the analysis plan. |
|
- * The required amount of DNA template depend on the length to be sequenced. The sample is used at 1–5μL per sequencing reaction. Assuming about 500 bases can be sequenced in one reaction, please provide as much sample as possible.
- * PCR products should be purified. Details on purification methods and optional sample purification services are provided.
- * We recommend agarose gel electrophoresis for concentration measurement and photos for reaction condition selection.
Related Service
Primer Walking is a method that designs sequencing primers from known sequences and reliably reads the sequence. However, since the readable sequence length in one sequencing is about 500 bases, the longer the target sequence, the more time, cost, and sample quantity are required.
In such cases, consider the “NGS Petit” small-scale analysis using next-generation sequencers. NGS Petit provides more efficient analysis and can deliver assembled contig data, just like Primer Walking.
Especially for analysis base lengths over 10kb, NGS Petit offers cost savings, so please consider using it.
