Primer design using NIH's Primer-BLAST tool.
Pedro C. Díaz, Francisco Franco García Calderon
Abstract
This protocol demonstrates the basic usage of the Primer-BLAST tool in order to design PCR primers from a given FASTA sequence.
The results should show the sequence of at least one pair of primers (forward and reverse) within the chosen parameters.
Steps
Go to NIH's Primer-BLAST tool
Open your internet browser (Google Chrome recommended).
Go to http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Enter FASTA sequence
Copy and paste your FASTA sequence in the text box or upload a FASTA file using the ¨Choose File¨ button.
You can select a custom range (i.e. the positional value of the first and last nucleotide of your ROI) for the forward and reverse primers.
Enter primer parameters
Enter the optimal parameters for your primers. Depending on your application, the PCR product size and the primer Melting temperatures are extremely important values!
Analyze
Click on the ¨Get Primers¨ button.
