Design Primers (Primer3): Testing Primers

You can use the primer design module to test primer sequences. You can test existing primer pairs to ensure that they match and produce the expected product. Alternatively, you can provide one primer and use the primer design module to design a suitable matching primer again for a specified region or sequence.

To test an existing primer pair

1. Open the sequence file you want to check your primers against.
2. Choose Analyze | Primers | Primer Design (Primer3) from the menu.
3. Check the Use this primer option for both primers, then paste or type the existing primer sequences into the appropriate Primer Sequences edit boxes.
4. The method drop-down list. will be automatically changed to Test Primers
5. Click OK.

Note that if you need to modify any parameters (for example increasing allowed mismatches) for testing a pair of primers please open the QuickTest Primer tool and use the Settings button.

Also note that when testing primer pairs the old Test PCR Primer Pairs output report is shown in the Results tab

To design a new primer to match an existing primer for a specified feature or region

1. Open the sequence file you want to analyze.
2. Select a feature of the sequence in the Map tab or the Features tab. Alternatively, select a region of the sequence in the Editor tab.
3. Choose Analyze | Primers | Primer Design (Primer3) from the menu.
4. Check the Use this primer option for the existing primer, then paste or type the existing primer sequence into the corresponding Primer Sequences edit box.
5. Ensure that the Find primer option is selected for the primer you want to design.
6. Ensure that Amplify Feature/Region is selected from the design method drop-down list, and that the feature you want to amplify is selected.
7. Click OK.

MacVector will test the existing primer, then design suitable matching primers to amplify the specified region. If you prefer to design a matching primer for any product size then use the next instructions instead:

To design a new primer to match an existing primer for any product size

1. Open the sequence file you want to analyze.
2. Choose Analyze | Primers | Primer Design (Primer3) from the menu.
3. Check the Use This primer option for the existing primer, then paste or type the existing primer sequence into the corresponding Primer Sequences edit box.
4. Ensure that the Find primer option is selected for the primer you want to design.
5. Select Region to Scan from the design method drop-down list.
6. Click OK.

By default this method will design a matching primer to produce products between 100 and 300 bases in length. However, you can change the minimum and maximum Product size values to design primers to produce shorter or longer products.

Related Topics.

Primers

Design Primers (Primer3): Advanced Options

Sequencing primers/probes

Primer Database

QuickTest Primers