Jun 21, 2025
Unmarked_Gene_Deletion_Ralstonia_Primer_Design
- 1Lowe-Power Lab, UC Davis
Protocol Citation: Noah Guillome 2025. Unmarked_Gene_Deletion_Ralstonia_Primer_Design. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1mrp2vr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 19, 2025
Last Modified: June 21, 2025
Protocol Integer ID: 220545
Keywords: Cloning, Primer Design, Primers, Unmarked Deletion, Ralstonia, pUFR80, Targeted Gene Knockout, NEBuilder, Primer-BLAST, Benchling, Gene Deletion, Homologous Recombination, Genome Editing, Chromosomal Integration, unmarked gene deletion in ralstonia, primer design step, unmarked gene deletion, target gene, primer design, design of primer pair, genome integration, genome, pufr80 vector backbone, primer pair, compatible with pufr80, primer, pufr80, plasmid construction, regulatory region, plasmid, gene
Abstract
This protocol describes the primer design step in generating an unmarked gene deletion in Ralstonia using the pUFR80 vector backbone. It guides the design of primer pairs to amplify ~500 bp upstream and downstream homologous regions with overhangs compatible with pUFR80 linearized by HindIII-HF and EcoRI-HF.
The goal is to generate primers that minimize off-targets, have self-complementarity scores ≤ 4 (and 3’ scores ≤ 2), and avoid amplifying the target gene while preserving nearby coding and regulatory regions. These primers are then used to build fragments for later steps in plasmid construction and genome integration.
Guidelines
This protocol is intended for generating an unmarked gene deletion in Ralstonia using the pUFR80 vector backbone. It assumes the use of NEBuilder for plasmid assembly and Benchling for sequence management. If working with a different organism, plasmid backbone, or assembly method, primer design parameters and workflow may need to be adjusted.
Materials
Computer with internet connection
Safety warnings
- In difficult sequence regions, it may be necessary to use primers with self-complementarity scores > 4 or 3′ scores > 2. These primers may still work, but they often have reduced efficiency and an increased risk of primer-dimer formation.
- Avoid using primer pairs with off-target products unless the unintended product is significantly larger than the expected product and can be excluded by setting a short elongation time during PCR that could not amplify the off-target product.
- For challenging regions where standard primer placement fails, refer to the troubleshooting comment in Steps 2 and 3. Ensure primer placement does not result in inclusion of coding regions from the neighboring genes in the sequence between the upstream and downstream fragments. This would result in their unintended partial deletion later during the cloning process.
Primer Design
Identify the Target Gene
- Extract a 10 kb sequence surrounding the gene of interest: 5 kb upstream and 5 kb downstream.
- If the gene is on the reverse strand (i.e., oriented to the left), reverse complement the sequence so that it reads left-to-right, with the upstream region on the left and downstream on the right.
Upstream Primers
- For the reverse primer, select the 5'->3' sequence 20 bp directly upstream of the gene of interest on the minus strand. (If the upstream gene overlaps with your gene of interest, use the last 20 bp of the upstream gene)
- Set the desired PCR product size to 400–600 bp.
- Under "Primer Pair Specificity Checking Parameters", select the "nt" database and set the Organism to match your strain. For example, if working with GMI1000, select:
Ralstonia pseudosolanacearum (taxid: 267608)
- Run the Primer-BLAST and select a primer pair with minimal off-targets and low complementary scores (see comments for troubleshooting information)
Downstream Primers
- For the forward primer, select the 5'->3' sequence 20 bp directly downstream of the gene of interest on the plus strand. (If the downstream gene overlaps with your gene of interest, use the first 20 bp of the downstream gene)
- Set the desired PCR product size to 400–600 bp.
- Under "Primer Pair Specificity Checking Parameters", select the "nt" database and set the Organism to match your strain. For example, if working with GMI1000, select:
Ralstonia pseudosolanacearum (taxid: 267608)
- Run the Primer-BLAST and select a primer pair with minimal off-targets and low complementary scores (see comments for troubleshooting information)
Experimental Plasmid Construction
Prepare the Vector Backbone
- In NEBuilder, select “Add New Fragment” and paste the pUFR80 sequence and check both the Vector and Circular options.
- For producing a linearized fragment, select Restriction Digest method.
- Set the 5’ flank enzyme to HindIII-HF and the 3’ flank enzyme to EcoRI-HF and add the fragment.
Add the Upstream Fragment
- In Benchling, identify the full sequence amplified by the upstream primer pair, based on flanking regions and primer pair generated via NCBI Primer-BLAST.
- Copy this amplified upstream sequence.
- In NEBuilder, select “Add New Fragment” and paste the sequence.
- For producing a linearized fragment, select PCR method and add the fragment.
Add the Downstream Fragment
- In Benchling, identify the full sequence amplified by the downstream primer pair, based on flanking regions and primer pair generated via NCBI Primer-BLAST.
- Copy this amplified downstream sequence.
- In NEBuilder, select “Add New Fragment” and paste the sequence.
- For producing a linearized fragment, select PCR method and add the fragment.
Finalize and Export
- Save the NEBuilder project and export the JSON file for documentation.
- In the project summary, locate the primer pairs designed for each fragment. NEBuilder will color-code primer binding regions to indicate homology.
- Use this summary to order the primers.
Archive the Assembled Plasmid
- Copy the entire assembled plasmid sequence from NEBuilder.
- Paste the sequence into Benchling and save it as a new plasmid entry.
- Run “Auto-Annotate” using the pUFR80 reference plasmid.
- Manually annotate the upstream and downstream fragments based on the inserted sequences. These annotations will support future alignment after plasmid sequencing.
External Primers
- Define a search range within Primer-BLAST: forward primer at least 100 bp before the upstream region and reverse primer at least 100 bp after the downstream region.
- Set the desired PCR product size to < 4500 bp.
- Under "Primer Pair Specificity Checking Parameters", select the "nt" database and set the Organism to match your strain. For example, if working with GMI1000, select:
Ralstonia pseudosolanacearum (taxid: 267608)
- Run the Primer-BLAST and select a primer pair with minimal off-targets and low complementary scores