Primer walking service is a useful method to obtain a sequence of size 2-10kb. For a bigger size or a large construct DNA, Psomagen recommends shotgun sequencing to save time and cost.
For primer walking, please keep in mind:
- The time for 1kb single contig varies and may take 5 to 6 days due to primer synthesis and sequencing failure
- Time can be saved when primers are synthesized bidirectionally
- A phred score of 20 or higher quality contig is required
- Results are based on the number of primers and sequence used for primer walking, the abi file used for primer walking, and the contig sequence and contig quality
With primer walking, starting with the end sequencing product by using the primer provided or designed, the internal primer will be designed and processed. When using a new primer but with the same template, the internal primer will continue reading at the appropriate location. In addition, each walking will require approximately 5 to 6 days at an enabling extension of 500bp to 600bp per direction.