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- Open the DNA sequence file that will be used as the template.
- Highlight the region to be amplified and click Analysis, Primer Design, Find PCR Primers.
- Click on More to see additional information about the primers. Generally, there will be oligo sequences in this window that need to be deleted (they are left over from the previous oligo design). If the oligos aren’t in the database, they may be entered at this time. Also, any parameters that you wish to change can be changed at this time.
- If the primers are already in the database, click on the box with the three dots next to the sense and find the sense primer in the database. Repeat with the antisense. Cut any non-template derived sequence from the 5’ end of each oligo and paste it into the box Attach to 5’ terminus.
- Click Apply.
- If the primers you designed are consistent with the chosen parameters and if they are appropriate for the template, the primers with be shown in the text pane with information about the PCR product size, the optimal annealing temperature, and other information about the primers including Tm, %GC, and so on. The primers can be checked for dimers, hairpins, and predicted duplex formation either in this screen, in the PCR design screen, or directly from the oligonucleotide database. Print this text screen because this information will not be saved in the database file. To save this information, the sequence file must be saved as a molecule file .
- The PCR product that is predicted to result from the primers shown can be saved in the database by clicking on the product description with the right mouse button and selecting Save to database. This is particularly useful for constructing recombinant molecules using this PCR product as the cloned insert.
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