Single or double strand sequencing by primer walking
Sequencing by primer walking allows rapid and precise sequence identification of long fragments with highest accuracy.
It starts using standard or specific primers to sequence the first 750 - 1000 bases of the delivered PCR product, plasmid or BAC, PAC, Cosmid, Fosmid DNA from one or both sides. Based on the resulting sequence data, we define and synthesise new primers which are used to sequence further bases thus “walking” along the sequence.
Single strand sequencing
The entire region of interest is sequenced single stranded. A final data accuracy of > 99.9% is guaranteed.
Double strand sequencing
De novo sequencing typically requires sequencing of both strands.
The region of interest is sequenced double stranded until both strands are completely deciphered. If there are any ambiguities in the sequence, further primers are designed to resolve them. This process ensures the highest data accuracy of 99.999% which represents less than one error in 100,000 base pairs.
Our Primer Walking Service includes:
- Quality control of incoming DNA
- Verification of the provided reference sequence
- Design and synthesis of specific primers
- Assembly of the sequence data
- Quality check of the consensus sequence incl. quality report
- Free storage of template DNA and all primers for 3 months
- A project report on material, method and results on request
- Delivery time: 2 kb within 6-8 working days
A reference sequence further speed up the project as this enables us to define multiple primers from the start.
Sending samples the right way for primer walking
Sending samples according to the requirements below helps us to do our job better and provides you with accurate results.
- Use 1.5 ml safe-lock tubes for your samples and primers
- Do not tape or wrap tubes with parafilm. Safe-lock tubes offer perfect sealing and evaporation protection
- Label your template tubes with our free Tube Labels
- Use a water resistant marker for any additional labelling of sample tubes
- Sending us a reference sequence speeds up project time and allows us to define multiple primers right from the start!
Use the following concentration and amount for your samples:
||Min 100 ng/µl
||Min 15 µl
||Min 10 ng/µl
||Min 15 µl
||Min 1 µg/kb
||Min 100 ng/kb
||Min 2 µg/kb
||Min 200 ng/kb
Optimum primer conditions:
- Primers must not contain phosphorylation or fluorescent dyes
- The optimum primer length is between 16-25 bases
- The primer melting temperature (Tm) should be 50 - 62°C
- The GC content of the primer should be 35-60%
- Ideally one G or C should be located at the 3' primer end
- The number of 3' Gs or Cs should not exceed 2 Gs or Cs
- If possible, avoid >3 identical bases in a row in the sequence
Primer concentration and volume:
- Exactly 10 pmol/µl primer concentration is required per sequencing reaction
- Each primer must have a total volume of 15 µl (double distilled water or 5mM Tris-HCl); 5 µl of primer volume is required for every additional sequencing reaction
- Concentration of primers with wobble bases must be calculated according to the following formula: nX x ConcPrimer