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Primer Walking

Ordering Inquiry

Sequencing data will be delivered at a low price by the rapid sequencing service utilizing our high quality oligo synthesis technology. All analyses “from PCR amplification to sequencing” are fully supported. We can be a dedicated sequencing department for your laboratory.

Primer Walking

Long sequences are determined by Primer Walking method with your provided DNA samples.
High quality data is available by continuous sequencing using primers we designed and synthesized from the result of the previous sequencing.

Service Description

Several sequencing analyses are performed with your provided DNA sample.
New primers are designed from the sequencing result and synthesized to be used for another sequencing reaction, hence long sequences are determined by repeating this process.

Please choose either from two methods, Single Strand Sequencing (SSS) and Double Strand Sequencing (DSS), depending on your application.

■The service includes■

– Quality check

– Primer design and synthesis

– Sequencing reaction

– Sequencer run

– Data check

– Assembled data generation

 

Reagent: BigDye Terminator v3.1

Equipment: 3730xl DNA Analyzer

 

  • * Averaged sequencing length per one reaction is approx. 500 bases. Sequencing will be repeatedly performed for several times according to the required sequencing length.
  • e.g.) For approx. 3.0kb primer walking (SSS), sequencing reaction will be normally performed 6 times, i.e. 3,000bp / 500bp (per reaction) = 6 times.

Deliverables

The report is to be sent via web site as well as by CD-R.

Deliverables Data recorded in CD-R
– HSS synthesized primer
– CD-R		 – Base sequence data (txt format)
– Waveform data (Ab1 format)
– Assembled data (txt format)
– Primer information
  • * Free of charge viewer software for waveform data (Ab1 file) is informed at the first order.
  • * If required, results for each sequencing reaction is to be reported by E-mail accordingly.
  • * Please note that the data uploaded on web site is to be deleted after approx. 3 months.

Price and Turnaround

Required number of sequencing differs depend on how many bases are ordered, so the price and the turnaround would vary accordingly.

The price includes design and synthesis of the primer to be used for Primer Walking, sequencing and assemble works.

(tax exclusive)
Base Length Single Strand (SSS) Double Strand (DSS)
Unit Price Turnaround Unit Price Turnaround
1.0kbp or less 22,000 approx. 2 weeks 44,000 approx. 2 weeks
1.5kbp or less 30,500 approx. 3 weeks 61,000 approx. 3 weeks
2.0kbp or less 39,000 78,000
2.5kbp or less 56,000 approx. 1 month 112,000 approx. 1.5 months
3.0kbp or less 64,500 129,000
3.5kbp or less 73,000 146,000
4.0kbp or less 81,500 163,000
5.0kbp or less 107,000 approx. between 1.5 and 2 months 214,000 approx. between 2 and 2.5 months
6.0kbp or less 124,000 248,000
7.0kbp or less 149,500 299,000
8.0kbp or less 166,500 333,000
9.0kbp or less 192,000 384,000
10.0kbp or less 234,500 469,000
  • * Lead-time may be varied depend on the number of samples or our sequencing schedule.
  • * Shorter turnaround may be available for known sequences. Please inquire.

Requirements

DNA sample Plasmid: Concentration; 100-300ng/uL, Quantity*1
PCR product: Concentration; 20ng/uL or more, Quantity*1,2
Primer (custom primer) For any kind of sample: Concentration; 10uM, Quantity; 10uL or more
Primer is used for the first sequencing.
(For free-of-charge universal primers, please select from here.)
Electrophoresis image Please send an image of a marker and your sample on the same gel with their quantity description.*3
Order form Please download the form from our web site.*4
  • *1 Required amount of DNA sample differs depend on the length to be sequenced.
    1-5 uL of a sample is required for one reaction to generate approx. 500 bases data.
    Please prepare for your sample as much as possible.
  • *2 Please make sure to purify PCR products after PCR reaction. For our sample purification service, please click here for details.
  • *3 Agarose electrophoresis is recommended for measuring sample concentration.
    Electrophoresis image sent with an order form will be used as a reference to select the reaction conditions. Please provide the marker name, apply amount of the marker and the sample on the electrophoresis image.
  • *4 Please select the primer walking plan from “(SSS)” or “(DSS)” on custom sequencing service order form.

Related Service: Small Scale Sequencing (NGS Petit)

Primer Walking method enables to reliably read the region you want to determine the sequence by designing sequencing primers based on known sequences, but each run covers only a limited length of the sequence such as 500 bases.
Therefore, the longer the length becomes, the more time, cost, and sample amount are required.

In such case, “NGS Petit” would be a good alternative. This is an efficient small scale sequencing service provided by next generation sequencer.
Delivery is also available in form of assembled contig data, just same as Primer Walking.

For 10kb or more, the price would be reasonable by using NGS Petit service. If you are interested, please inquire.

For details of small scale sequencing, please inquire.

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