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Troubleshooting Your Design:

Here are a few tips to help troubleshoot your design:

  1. After downloading your results, you may find that some targets have no designed primers.
  2. Check the 'Error' tab in the Excel file and adjust your design parameters based on the error. Here are some examples:
    • Error on left or right primer: considered 906, GC content failed 830, low Tm 76, ok 0
      • 'considered 906': 906 oligos on this side of the target were evaluated for the design.
      • 'GC content failed 830': 830 of these oligos had insufficient GC content (below 40%), indicating a challenging region for design.
      • 'low Tm 76': 76 of these oligos had a Tm too low to meet the design criteria.
      • 'ok 0': None of the oligos were retained on this side of the design.
    • Troubleshooting: In these cases, you can either adjust the size range of your design to target a different region or lower the expected Ta to allow for more flexibility in the design.

      • Note: If you encounter other errors, feel free to reach out at mathilde.delpeuch@gmail.com.
      I'm happy to assist! :)

PrimDesign

Designing Oligos with Precision and Ease

Design for CRISPR application:

PrimDesign designs two types of primer pairs:

  • PCR primers: Amplify ~1000 bp surrounding the cut site to detect potential InDels
  • Sequencing primers: Generate a shorter amplicon closer to the cut site for precise sequencing
Included QC: Primers are BLASTed againt gDNA to ensure specificity and meet the standard quality requirements for optimal PCR reaction

How to:

  1. Download the excel template (see below)
  2. Fill it up with : Target name, gRNA sequence(s) without PAM and gRNA genomic location, if available
    3. Note: adding the location helps with difficult designs such as alt chromosome, repeated area, etc (e.g. chr17:46,024,163-46,024,182)
    New: You can also input multiple gRNA sequences for a single target by entering them in the same excel cell, separated by spaces.
  3. Enter your parameters:
    4. - Directly on the webpage: Parameters entered here will apply to all targets in your Excel list
    - Or in the Excel file: You can customize parameters for specific targets by filling in the desired values directly in the corresponding cells.
    For example, if you want a Tm of 60°C for target_1, enter "60" in its cell. Any cells left empty will use the default values specified in the webpage form, ensuring only the customized target is computed with the specified value.
  4. Upload the Excel file and click Submit

Parameters:

Design for qPCR application:

Principle: Based on the Ensembl ID, PrimDesign will fetch the coding sequence of the gene and find the exon-exon junction's positions
Then primers are designed to amplify the sequence surrounding each junction.
Included QC: Primers are BLASTed againt gDNA to ensure specificity and meet the standard quality requirements for optimal qPCR reactions.

How to:

  1. Download the excel template (see below)
  2. Fill it up with: Target name and reference ID from the database: e!ENSEMBL
    (e.g. ENST00000301071)
  3. Enter your parameters:
    4. - Directly on the webpage: Parameters entered here will apply to all targets in your Excel list
    - Or in the Excel file: You can customize parameters for specific targets by filling in the desired values directly in the corresponding cells.
    For example, if you want a Tm of 60°C for target_1, enter "60" in its cell. Any cells left empty will use the default values specified in the webpage form, ensuring only the customized target is computed with the specified value.
  4. Upload your file and click Submit

Parameters:


Design for a custom sequence:

PrimDesign can design primer pairs based on a provided sequence:

  • Primers are tailored to amplify the target region of interest
  • Primers meet the standard quality requirements for PCR applications

How to:

  1. Enter the target name
  2. Provide the template sequence by filling up the form on the right
  3. Enter the desired parameters
  4. Click Submit

Parameters:


Design for genotyping:

PrimDesign can create sets of primer pairs to genotype a long input using multiple PCR reactions:
  • Primers are designed to amplify the target by segments of 1000bp each
    • Note : If another product size is desired, fill up the max and min in the form
  • Primers meet the standard quality requirements for PCR applications
  • Amplified segments overlaps, to allows sequencing of the entire area without breaks
    • Note: The overlap is calculated as 3% of the segment size but can be customized using the form

How to:

  1. Provide the target name and the template sequence in the form on the right
  2. Choose the type of sequence : circular (e.g. plasmid) or linear (e.g.long sequence, ssODN,...)
  3. Enter the desired parameters
  4. Click Submit

Parameters: