Primer Walking
We provide single directional and bi-directional primer walking of DNA constructs. Bi-directional primer walking is recommended for more accurate results. Below are the sample requirements: -
| Length | Requirements | |
|---|---|---|
| < 3kb Plasmid | 50-200 ng/µl of purified plasmid in 50 µl dH2O | 1.5kb per week |
| > 3kb Plasmid | Whole bacterial colony in a freshly streaked LB agar plate with appropriate antibiotics (free plasmid extraction and purification included) | |
1) Bi-directional Primer Walk
For bi-directional primer walking, DNA sequencing will start with two universal primers and is continued using primers derived from the ends of the newly determined DNA sequences until the whole insert/ fragment is fully sequenced on both ends. If there are any ambiguities in the sequence, we will design new primers to resolve them.
Figure 1: Bi-directional primer walking
of construct provides 99.9% accuracy with a double
confirmation of the insert sequence on both
strands.
2) Single Pass Primer Walk
For single pass primer walking, DNA sequencing also starts with two universal primers and continues using primers derived from the ends of the newly determined DNA sequences until the overlapping region of both strands are determined. Single Pass Primer Walking is suitable for the sequencing of constructs with a known insert sequence.
Figure 2: Single pass primer walking of constructs provides a faster turnaround rate with 98.5% accuracy of the insert sequence.
Primer Walking Service Includes:
- Primer design & synthesis (Primers will be delivered to you at the end of the project)
- Sequencing reactions
- Data assembly and sequence aligning to generate the consensus sequence