PCR Primer Design

PCR PRIMER DESIGN

Create primer pairs for PCR matching your experimental conditions with easy-to-use primer design software.

Use SeqBuilder Pro to design PCR primers you can rely on to work the first time.

Let’s be honest, there is nothing more discouraging than spending the time and money setting up a PCR reaction only to have it fail multiple times. Though many factors can impact the success of PCR, often when failure happens, poor PCR primer design is the culprit. SeqBuilder Pro eliminates this problem by designing PCR primers matching your unique experimental conditions and giving you the ability to see the impact of potential changes before you try them out in the lab. A perfect balance of guidance, editability, and customization, SeqBuilder Pro enables you to design PCR primers that you can rely on to work the first time.

Create PCR primers fast in 4 simple steps

Step 1

Select feature or range you want to amplify

Step 2

Select Priming > Create Primer Pairs

Step 3

Adjust parameters, or accept the defaults

Step 4

View and analyze results!

Learn more about PCR Primer Design

Resources | Tutorials | FAQs | Citations | User Guide

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Resources

Please see our resources below for more information on PCR primer design.

Learn SeqBuilder Pro Workflows

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Master Our Foundation Application, SeqBuilder Pro

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SeqBuilder Pro FAQ: Answers to Your Webinar Questions

Read Blog Post

How to perform PCR site-directed mutagenesis using Lasergene’s protein and primer design tools

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Structure-guided molecular cloning for improving site-directed mutagenesis and stability in protein design

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Cloning and Primer Design Workflows Webinar

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Enhanced Sequence Analysis for Today’s Molecular Biologist

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Lasergene Molecular Biology Overview

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Tutorials

Watch one of our videos or check out one of our written tutorials to learn more about using the PCR primer design tool in SeqBuilder Pro.

Primer Design in SeqBuilder Pro

In this video, we show you how to design and modify PCR primers in SeqBuilder Pro.

Using Primer Catalogs in SeqBuilder Pro

SeqBuilder Pro enables you to easily import and export groups of primers for use across multiple projects through the creation of primer catalogs.

Modifying Primers in SeqBuilder Pro

Learn how to modify PCR primers created using SeqBuilder Pro.

Extracting PCR Product for Cloning in SeqBuilder Pro

In this video, learn how to use SeqBuilder Pro to extract PCR product to use in virtual cloning simulations.

FAQs

During primer design, how does SeqBuilder Pro calculate Tm?

In its role as a PCR primer design tool, SeqBuilder Pro uses the nearest neighbor model to measure the stability of a given duplex. It is based upon the free energy of adjacent dinucleotides and…

In its role as a PCR primer design tool, SeqBuilder Pro uses the nearest neighbor model to measure the stability of a given duplex. It is based upon the free energy of adjacent dinucleotides and calculated from dinucleotide entropy and enthalpy data presented in Bresslauer et al. for DNA, and Freier et al. for RNA.

How do I export primers?

Choose File > Save Primer Catalog and choose from any of three formats: tab-delimited text (.txt), FASTA (.fas), or primer catalog (.pri).

Can I import primers into a new project?

After doing primer design, save your primers as a primer catalog (.pri). In the new project, use File > Import Primers from a Catalog to open the saved catalog. SeqBuilder Pro will then search your sequence for priming sites.

Can I add restriction sites to my primers?

Several SeqBuilder Pro views allow you to apply and view the locations of restriction sites on a sequence. In addition, you can clone your insert into a vector via restriction cloning.

How can I add mutations via primers?

In SeqBuilder Pro, you can mutate primers in the Primer Design view manually; or automatically using the Introduce Codon Change/Mutation tool. Alternatively, use Protean 3D’s protein design …

In SeqBuilder Pro, you can mutate primers in the Primer Design view manually; or automatically using the Introduce Codon Change/Mutation tool. Alternatively, use Protean 3D’s protein design tools to predict suitable mutation sites for PCR-directed mutagenesis.

How do I know if my PCR primers are stable?

This is shown in two areas of the Primer Design view

The “Mispriming” section shows primer binding sites and the most stable conformation of dimers,…

This is shown in two areas of the Primer Design view.

The “Mispriming” section shows primer binding sites and the most stable conformation of dimers, pair dimers, and hairpins, if any exist. SeqBuilder Pro flags primer pairs if the final pentamer value of their dimer exceeds the 3’ pentamer stability threshold defined in the Primer Characteristics settings.

The “Alternate Pairs” section shows quality scores for each primer pair that SeqBuilder Pro identifies.

What should the target Tm be for my PCR primers?

For best results, PCR primer pairs should have closely matched melting temperatures to promote amplification of both strands in equal amounts. Use the Primer Characteristics dialog (Priming > Create Primer Pairs) to adjust the default target Tm. SeqBuilder Pro will attempt to locate primers as close to this value as possible.

How can I adjust the length of my PCR primers?

Before SeqBuilder Pro creates your PCR primers, you can define the target min/max primer length in the Primer Characteristics dialog (Priming > Create Primer Pairs). Following primer pair creation, you can adjust the length from within the Primer Design view by dragging the “handles” at either end of the primer.

How do I create PCR primers at the right location?

You can use the Locations dialog (Priming > Create Primer Pairs) to define where SeqBuilder Pro will search for primers. Select Primers end exactly…

You can use the Locations dialog (Priming > Create Primer Pairs) to define where SeqBuilder Pro will search for primers. Select Primers end exactly at selection to search for primers that lie exactly within the selected region. Select Choose optimal primer location to search for a specified maximum number of primers that begin and end within the distance specified outside of the selected region.

Can I create a new primer pair from two existing primers?

Yes, this can be done easily when you have SeqBuilder Pro as your primer design software. Select the two primers you wish to pair from the Primers or the Primer Design views. Then choose Priming > Selected Primers. A new primer pair will be created from the two selected primers and becomes the active pair.

Citations

Fungal Isocyanide Synthases and Xanthocillin Biosynthesis in Aspergillus fumigatus
Fang Yun Lim, Tae Hyung Won, Nicholas Raffa, Joshua A. Baccile, Jen Wisecaver, Antonis Rokas, Frank C. Schroeder, Nancy P. Keller. mBio May 2018, 9 (3) e00785-18; DOI: 10.1128/mBio.00785-18.

Development of a synthetic gene network to modulate gene expression by mechanical forces.
Kis, Z. et al. Sci. Rep. 6, 29643; doi: 10.1038/srep29643 (2016).

Genome-based population structure analysis of the strawberry plant pathogen Xanthomonas fragariae reveals two distinct groups that evolved independently before its species description.
Michael Gétaz, Marjon Krijger, Fabio Rezzonico, Theo H. M. Smits, Jan M. van der Wolf, Joël F. Pothier. Microb Genom. 2018 Jul; 4(7): e000189. Published online 2018 Jun 6. doi: 10.1099/mgen.0.000189.

A putative, novel coli surface antigen 8B (CS8B) of enterotoxigenic Escherichia coli.
Samuel M. Njoroge, Christine J. Boinett, Laure F. Madé, Tom T. Ouko, Eric M. Fèvre, Nicholas R. Thomson, Samuel Kariuki. Pathogens and Disease, Volume 73, Issue 7, October 2015, ftv047, https://doi.org/10.1093/femspd/ftv047.

Accurate, simple, and inexpensive assays to diagnose F8 gene inversion mutations in hemophilia A patients and carriers.
Debargh Dutta, Devi Gunasekera, Margaret V. Ragni, Kathleen P. Pratt. Blood Adv 2016; 1 (3): 231–239.

Functional Consequences of CHRNA7 Copy-Number Alterations in Induced Pluripotent Stem Cells and Neural Progenitor Cells
Gillentine, Madelyn A. et al. The American Journal of Human Genetics, Volume 101, Issue 6, 874 – 887.

K13 Propeller Alleles, mdr1 Polymorphism, and Drug Effectiveness at Day 3 after Artemether-Lumefantrine Treatment for Plasmodium falciparum Malaria in Colombia, 2014-2015.
Madeline Montenegro, Aaron T. Neal, Maritza Posada, Briegel De las Salas, Tatiana M. Lopera-Mesa, Rick M. Fairhurst, Alberto Tobon-Castaño. Antimicrobial Agents and Chemotherapy Nov 2017, 61 (12) e01036-17; DOI: 10.1128/AAC.01036-17.

TIA: algorithms for development of identity-linked SNP islands for analysis by massively parallel DNA sequencing.
Farris, M.H., Scott, A.R., Texter, P.A. et al. BMC Bioinformatics 19, 126 (2018) doi:10.1186/s12859-018-2133-2.

Ethanol Upregulates NMDA Receptor Subunit Gene Expression in Human Embryonic Stem Cell-Derived Cortical Neurons.
9. Xiang Y, Kim K-Y, Gelernter J, Park I-H, Zhang H (2015). PLoS ONE 10(8): e0134907.

Identification of novel alleles of the rice blast resistance gene Pi54.
Vasudevan, K. et al. Sci. Rep. 5, 15678; doi: 10.1038/srep15678 (2015).

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The best product I’ve found

“I have used DNASTAR for developing primers that will work with SNPs. I tried other tools, but I haven’t found anything that I like more. There are so many different parts of the software that I like and that I use regularly when I design primers. It’s really been the best product that I’ve found. It is user friendly. It is also extremely helpful to map out all the regions on the sequence when designing primers.”

Blaire Bacher, Orion Genomics
Every bit the match of Vector NTI

“I’ve found the package to be excellent and exemplary. In particular, the SeqBuilder module is very versatile and every bit the match of Vector NTI.”

Dr. Chris Winefield, Lincoln University
Easy

“Easy primer design.”

Dylan MacGregor Johnson, University of Louisville

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