Issue
When attempting to design primers using Benchling’s Primer Wizard for longer genes from various genomes, users frequently encounter delays, errors, or failed primer generation. This issue arises particularly when selecting a target gene within the full genome and specifying a maximum amplicon size larger than the gene itself (e.g. 400bp larger). The same parameters used in Primer3 successfully generate primers without issues.
Environment
Product: Molecular Biology
Feature: Primer Wizard
Resolution
1. Reduce the Target Region:
- Instead of selecting the entire gene, include only a region approximately 1kb larger than the target gene when setting up the primer design.
- This can be done by creating a new sequence from the genome that includes the gene and flanking regions.
- Use this smaller sequence as the input for the Primer Wizard.
2. Use Advanced Region Settings:
- Utilize the “Included Region” feature in the Primer Wizard.
- Set this included region to encompass the gene plus a small flanking area. This will help Benchling handle the primer design process more efficiently without needing to create a separate sequence.
3. Monitor Amplicon Size:
- Ensure the amplicon length fits within Benchling’s default amplicon length range (200 - 1000bp). If necessary, adjust this range to accommodate larger amplicons.
Cause (Optional)
The issue appears to be related to the large size of the genome. The Primer Wizard struggles to process large sequences efficiently, particularly when a large region or the entire genome is selected as the target area.