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If you set "5' backbone length", then the number of bases you set from 5' end of the backbone sequence will be checked for unwanted connections. The same about "3' backbone length", but from the 3' end. If the checked end has hairpins, self- and homo-dimers you will see the following dialog:

Children Display
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Type two primers for running In Silico PCR. If the primers pair is invalid for running the PCR process then the warning is shown. Also, primers for the running In silico PCR can be chosen from a primer library. Click the following button to choose a primer from the primers library: 

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  <img src="/wiki/download/attachments/13435073/Primers Library.png"/>
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 The following dialog will appear:  

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  <img src="/wiki/download/attachments/16125631/In Silico PCR_2.png"/>
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The table consists of the following columns: name, GC-content (%), Tm, Length (bp) and sequence. Select primer in the table and click the Choose button. 

Click the Reverse-complement button for making a primer sequence reverse-complement: 

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  <img src="/wiki/download/attachments/13435069/In Silico PCR_1.png"/>
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Click Show primers details for seeing statistic details about primers. 

When you run the process, the predicted PCR products appear in the products table.

Products table

There are three columns in the table:

• region of product in the sequence
• product length
• preferred annealing temperature 

 Click the product for navigating to its region in the sequence. 

 Click the Extract product(s) button for exporting a product(s) in a file or use double click for that. 

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Other sequences in PCR reactions

Sequences which are supposed to be in the reaction mixture. It's important primers do not have unwanted connections to these sequences, so it will be checked that there are no hairpins, self- or hetero-dimers between the result primer and any sequence from the "Other sequences in PCR reaction" file.