DNASTAR

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 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 workflow 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, 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 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.