Many real time PCR techniques use a third oligonucleotide that will anneal inside the desired product. This makes is possible to track the progress of the PCR amplification using one of the many proprietary technologies available. The primer design module enables you to design these. You can design an internal primer suitable for use with a pair of existing primers, design external primers to suit an existing internal primer, or design all three primers from scratch.
To design a pair of external primers and a suitable internal hybridization primer from scratch.
1. Open the sequence file you want to analyze.
2. Select a feature of the sequence in the Map tab or the Features tab that you want to monitor. Alternatively, select a region of the sequence in the Editor tab. By default, primers are chosen from a 200 bp long region on either side of the feature or region selected.
3. Choose Analyze | Primers | Primer Design (Primer3) from the menu.
Note. When the Primer Design (Primer3) dialog box opens it is populated with information about the feature or region you selected in the active sequence window. You can populate the dialog box with information about an alternative feature in the active sequence using the feature selector drop-down menu, or you can specify an alternative region by providing residue numbers manually.4. Ensure that Amplify Feature/Region is selected from the design method drop-down list.
5. Make sure Find Primers is selected for both primers.
6. Select the Hybridizing Primer Sequence check box.
7. Make sure Find Hybridizing Primer is selected.
8. Click OK.
Please note that you may also use Flanking Regions at step 4 if you require the primers to be chosen from more specific areas than the default 200bp regions.
1. Open the sequence file you want to check your primers against.
1. Choose Analyze | Primers | Primer Design (Primer3) from the menu.
2. Check the Use this primer option for both primers, then paste or type the existing primer sequences into the appropriate Primer Sequences edit boxes.
3. Select Region to Scan from the design method drop-down list.
4. Make sure the region to scan entirely encompasses the area that contains your expected product. If in doubt change to the entire sequence.
5. Ensure the product size is changed to suitably relaxed parameters. e.g. between 0.5kb below and 0.5kb above the product size you would expect.
6. Select the Hybridizing Primer Sequence check box
7. Make sure Find Hybridizing Primer is selected.
You should consult the manual of the hybridization primer technology that you are using for specific values to use for this primer. Make sure that the settings in the Hybridization Primer advanced parameters tab are set to these.8. Click OK.
1. Open the sequence file you want to analyze.
2. Select a feature of the sequence in the Map tab or the Features tab that you want to monitor. Alternatively, select a region of the sequence in the Editor tab.
By default, primers are chosen from a 200 bp long region on either side of the feature or region selected.3. Choose Analyze | Primers | Primer Design (Primer3) from the menu.
Note. When the Primer Design (Primer3) dialog box opens it is populated with information about the feature or region you selected in the active sequence window. You can populate the dialog box with information about an alternative feature in the active sequence using the feature selector drop-down menu, or you can specify an alternative region by providing residue numbers manually.4. Ensure that Amplify Feature/Region is selected from the design method drop-down list.
5. Select the Hybridizing Primer Sequence check box.
6. Make sure Find Hybridizing Primer is selected.
7. Check the Use this primer option for Hybridizing Primer then paste or type the existing primer sequence into the appropriate edit box.
8. Click OK.